所谓肺硬化性血管瘤中的两种主要细胞的基因表达差异及意义
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摘要
前言
肺硬化性血管瘤(pulmonary sclerosing hemangioma)是由Liebow和Hulbell首先报道的肺部较为少见肿瘤,约占肺肿瘤的2-3%,最初认为是黄色瘤或错构瘤,长期以来被归类于肺的炎性假瘤范畴。随着时间的推移,人们逐渐认识到:肺的硬化性血管瘤并不是肺的炎性假瘤,而是一种实实在在的“良性”真性肿瘤,尽管其发病率较低,但由于临床CT、磁共振等影像学检查极易与肺癌相混淆,更为严重的是术中冰冻病理诊断又极易误诊为肺腺癌、细支气管肺泡癌以及类癌等,因而手术切除常常过度,给患者造成损害,所以对该肿瘤的研究和诊断备受重视。在我们的研究中,为了区分该肿瘤并不是炎性假瘤,也为了将来能够有一个更适合该肿瘤的名字。我们将该肿瘤称之为“所谓的肺硬化性血管瘤”(So-calledpulmonary sclerosing hemangioma,PSH)。由于该肿瘤一般呈良性临床经过,与其相平行的名称还有:良性乳头状神经内分泌肿瘤,良性肺泡细胞瘤,良性肺腺瘤,错构瘤等。之所以出现这些名称,是因为到目前为止PSH的起源尚不清楚。有人认为来源于内皮细胞、间皮细胞、肺泡上皮细胞、神经内分泌细胞等,最近还有人提出是来源于多潜能的原始呼吸道上皮细胞的观点。在新版的WHO分类当中,也只好将该肿瘤暂时放入“未定类肿瘤”当中。有人还对该肿瘤当中两种主要形态不同的细胞是否为同一起源提出了质疑,并观察到部分肿瘤术后若干年出现了复发和转移的现象。
针对PSH的分类困难、影像和病理诊断困难、起源不明、生物学特性不清的特点,我们收集了40余例的PSH资料,利用免疫组化、激光捕获微切割(LCM)、RT-PCR、单链构象多态性分析和DNA测序分析等方法,对该肿瘤进行系统深入的研究,期望找到能正确诊断该肿瘤的方法,并对其起源及生物学行为方面作以探讨。
材料与方法
1、材料
收集中国医科大学第一临床学院1995～2004年间病理诊断为PSH及手术切除病变旁正常肺组织蜡块标本40例。选其中17例女性,2例男性具有完整临床资料的病例进行基因表达方面的研究。该19例当中,12例无自觉症状,为体检时发现;7例有症状者分别表现为胸痛、咳嗽、痰中带血。胸部X线片或胸部CT显示均为孤立性肿块,多位于肺的周边,呈圆形、卵圆形高密度影,边缘光滑,无毛刺及分叶状改变。肿块位于右肺11例,左肺8例。未发现多发病灶及转移病例。发病时间为15天至5年不等。
2、免疫组化
选用单克隆抗体细胞角蛋白(cytokeratin L,CK-L)、上皮膜抗原(epithelialmembrane antigen,EMA)标记上皮性抗原,SP-B和TTF-1标记原始呼吸道上皮细胞或肺泡Ⅱ型细胞,波形蛋白(vimentin,VT)标记间叶来源细胞,CD34标记内皮来源细胞,CD68标记巨噬细胞,神经元特异性烯醇化酶(neuron specific endolase,NSE)和突触素(synaptophysin,Syn)标记神经内分泌来源的细胞以及单克隆P53抗体(DO-7)。上述即用型抗体及S-P超敏试剂盒均购自福州迈新物技术公司。将蜡块标本制成4μm连续切片,经脱蜡、脱苯、水化后进行HE和链菌素抗生物素蛋白-过氧化物酶(S-P法)免疫组化染色。以已知阳性组织为阳性对照,PBS缓冲液代替一抗为阴性对照。每张切片随机选取5个高倍视野,每高倍视野计数100个瘤细胞,无着色及阳性细胞数≤5%为(-),阳性细胞数＞5%为(+)。CK-L、SP-B、Vimentin、CD34、CD68、NSE、Syn阳性表达的判断为单纯胞质表达;EMA阳性表达的判断为膜/质表达;TTF-1、p53阳性表达的判断为单纯核表达或核、胞质同时表达。
3、激光捕获目的细胞、提取总RNA
取病变组织蜡块,制成6μm连续切片,Mayer's苏木素—伊红染色,二甲苯透明,切片晾干,放置在激光捕获显微切割(lasercapture microdissection,LCM)系统(LM200型)的倒置显微镜的载物台上。将附有一层乙烯乙酸乙烯酯薄膜(et hylenevinylacetate polymer,EVA)的扁平塑料帽紧密覆盖在切片的表面。定位光束标定被选细胞,发射激光束,分别捕获PSH组织中表面立方细胞和多角形细胞。每份标本分别捕获5000～10000个目的细胞。按照PicoPure~(TM) RNA Isolation Kit说明书操作提取总RNA(Arcturus Bioscience,Mt.View,CA)。
4、RT-PCR
按照RiboAmp(?)HS RNA Amplification Kit说明书操作。PCR扩增产物使用1.5%琼脂糖,100V电泳35min,溴化乙啶(EB)染色,凝胶成像系统(BioImageingSystem,UVP,CA)下观察。以β-actin为内对照。
5、DNA提取、P53基因PCR扩增、SSCP及测序分析
将捕获的PSH组织中表面立方细胞和多角形细胞加入预先加有50μL DNA裂解液(10 mmol/L Tris HCl,pH8.0;1 mmol/L EDTA PH8.0;1%Tween 20;200 mg/L蛋白酶K)的0.5 mL离心管上。48℃水浴14 h,95℃10 min灭活蛋白酶K,-20℃保存备用。分别取多角形细胞和立方细胞的DNA模板各10μL于50μLPCR反应体系进行P53基因(5～8外显子)PCR扩增。取6μL PCR产物加等量变性上样缓冲液混匀后,100℃热变性10 min后迅速置于冰水浴中冷却5 min,然后上样于10%非变性聚丙烯酰胺凝胶(49∶1)中;每组均以病变旁正常肺组织作对照。恒温电泳。凝胶经硝酸银染色。判断标准:待测样品的电泳带与正常对照的电泳带相比,若出现条带的增加、减少或位置的移动,即说明该样品存在着基因突变。同时取剩余的p53基因(5～8外显子)扩增产物(40μL左右)进行测序分析(上海联合基因测序部)。
结果
1、组织学改变
PSH组织形态多样,由实性区、乳头区、血管瘤样区和硬化区四种基本结构构成,并有不同程度的相互移行。肿瘤细胞主要有两种:一种是位于实性区和乳头区内的多角形瘤细胞,胞浆淡染或嗜酸,核圆或卵圆形,有小核仁,罕见或无核分裂像。另一种是被覆在乳头表面、血管瘤样区间隙及实性区裂隙内的细胞,多呈矮立方状,形态与肺泡Ⅱ型细胞极为相似,可融合成多核巨细胞,但无异型性。间质内可见较多的肥大细胞和以淋巴细胞为主的各种炎性细胞浸润,还可见含铁血黄素沉积、钙化和骨化等。多核瘤巨细胞和硬化性乳头的出现,是造成诊断困难的原因之一。
2、免疫组化结果
在40例PSH标本中,CK-L、EMA和SP-B只表达在表面立方上皮细胞或实性区内陷的立方上皮细胞和血管瘤样区的内衬上皮细胞,而间质多角形细胞为阴性;Vimentin则只表达于间质的多角形细胞;TTF-1在两种细胞内均有表达;CD34只在血管内皮细胞表达,在两种细胞内均无表达;多核瘤巨细胞则表达CK-L,SP-B和TTF-1,而CD68为阴性。此外,间质多角形细胞少量表达Syn和NSE。19例PSH组织中,3例(15.8%)标本p53蛋白呈阳性表达,且多角形细胞的表达强度高于表面立方细胞的表达;15例(78.9%)p53蛋白呈阴性表达,其中1例标本虽有p53蛋白呈阳性反应的细胞,但阳性细胞数≤5%。
3、RT-PCR结果
应用LCM方法,分别提取19例PSH当中的两种细胞的mRNA,RT-PCR扩增电泳后,所谓肺硬化性血管瘤组织中的两种细胞,其基因表达有着明显的区别,表面立方细胞有明确的CK、EMA、SP-B和TTF-1的mRNA表达,而间质内的多角形细胞有明确的Vimentin、Syn和TTF-1的表达,同时有弱的EMA的表达。两种细胞都未见Ch-A的表达。这一结果与免疫组化的蛋白表达结果极为相似。
4、SSCP及测序分析
SSCP法检测19例PSH组织P53基因突变,3例(15.8%)标本出现异常移动带,1例发生在第6外显子上,2例发生在第7外显子上,第5及第8外显子未见异常;在这3例突变中,单一多角形细胞突变2例,立方细胞和多角形细胞同时突变1例。DNA测序分析结果显示19例PSH组织中,P53基因的突变率为15.8%(3/19);单一多角形细胞突变2例,多角形细胞和立方细胞同时突变1例;1例发生在第6外显子;2例发生在第7外显子,第5、8外显子未见突变;3例均为错义突变。
结论
1、PSH组织当中的多核瘤巨细胞是表面立方细胞的融合,多核瘤巨细胞和硬化性乳头的出现,是造成病理诊断困难的主要原因之一。
2、PSH组织中的多角形细胞和表面立方细胞在形态表型上的差异可能是由于所处不同的分化状态所致,表面立方细胞趋向肺泡Ⅱ型细胞分化,而间质内的多角形细胞则具有多向分化的潜能。
3、PSH中两种细胞均可有突变型P53蛋白表达;多角形细胞中突变型p53蛋白表达率明显高于立方细胞;PSH中两种细胞均存在DNA错义突变,且间质多角形细胞突变高于表面立方细胞;P53蛋白表达与基因突变检测结果一致,提示PSH具有潜在恶性的可能。
Objective
Pulmonary sclerosing hemangioma(PSH) is an uncommon tumor of the lung and it s histogenesis and origin are uncertain to date.PSH consists of two major tumor cells: polyangular cells and cuboidal cells.Polyangular cells are true tumor cells which derive from primitive respiratory epithelium,but it remains controversial over whethere the surface lining cuboidal cells of PSH are true tumor cells or the results of responsive proliferation in the tumor genesis and whethere this two major tumor cells have the same origin.A general consensus appears to have been reached that PSH is a benign neoplasm,but a few PSHs are found to invade and metastasize.The phenomenon which lesion tissues invaded bronchus and peripheral mesenchyma can be seen reported.In order to further veritfy the origin and the possible character of PSH,we captured two major cells of PSH by laser capture micro-dissection(LCM).We selectedthe P53 gene which was related to pathogenesis of this disease as the subject of study and used immunohistochemical method,laser capture micro-dissection(LCM) technology,Single-stranded conformation polymorphism(SSCP) and DNA sequencing analysis to examine the expression of P53 protein and mutation of P53 gene in polyangular cells and cuboidal cells in PSH and valued its significance,providing reference to histogenesis and biological conduct of PSH.The expression of p53 protein and mutation of p53 gene are significant parameters which can reflect biological behavior of tumor cells.The aim of this study is to investigate the p53 protein expression and p53 gene mutation.Disclosing the different gene expression of the two cell types will provide the basis for the study of histogenesis of PSH tissue and helpful for differential diagnosis.
Materials and Methods
1、Patients
Paraffin-embedded PSH samples and normal lung tissue adjacent to PSH were obtained from 40 patients who were diagnosed with PSH at the First Clinical College of China Medical University between 1995 and 2004.Among those cases,19 cases were celected which had whole clinic data to do gene expression analysis.This study was conducted according to the regulations of the institutional review boards(China Medical University).The age range was 24-46 years,and the mean age was 34.5 years. Of the 19 patients,12 had no symptoms and their PSH was found upon routine examination;the other seven patients presented with symptoms including chest pain, cough and bloody phlegm.X Rays or CT scans revealed unitary masses,frequently in the periphery of the lungs.These masses were round or oval and of high density,with a smooth boundary,and with no burs or lobules.The masses were located in the right lung in 11 cases and the left lung in eight cases.The follow-up time(after surgery,to December 2005) ranged from 13-129 months;all patients survived,without recurrence or metastasis.
2、Immunohistochemistry
Low molecular weight cytokeratin(CK-L) and epithelial membrane antigen (EMA) sign epithelia antigen,surfactant protein B(SP-B) and thyroid transcription factor- 1(TTF-1) sign respiratory multipotential primitive epithelium or pneumocytes typeⅡ,Vimentin(VT) signs mesenchymal antigen,CD34 signs endothelial cells of vessels,CD68 signs macrophage,neuron specific endolase(NSE) and chromograninA(ChA) sign endocrine cell.Formalin-fixed paraffin-embedded tissue blocks were cut into 4 mm sections,de-waxed and hydrated.Immunostaining was performed with the streptavidin peroxidase system(Ultrasensitive;MaiXin,Fuzhou, China) according to manufacturer's instructions.The sections were incubated with a primary antibody(dilution 1:50;Santa Cruz Biotechnology,Santa Cruz,California, USA).Biotinylated goat anti-mouse serum IgG was used as a secondary antibody. After washing three times in phosphate-buffered saline(PBS),the sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase,and visualised by demonstration of conjugated peroxidase with diaminobenzidine as the substrate.The sections were counterstained with haematoxylin.For the negative control, primary antibodies were replaced with PBS;for the positive control,known positive tissue was used.A slide was considered negative or positive according to the absence or presence of positive staining:no staining or fewer than 5%of total cells positive for p53 was considered negative;greater than 5%of total cells positive for p53 was considered positive staining.
3、Laser capture microdissection of target cells,and extraction of RNA
The paraffin-embedded samples were sectioned successively at a thickness of 6 mm.The sections were subjected to Mayer's H&E staining,dehydration with gradient alcohol,and lucidification with xylene(for 5 min).After drying,the sections were placed onto the object stage of an inverted microscope connected to a laser capture microdissection system(model LM200;Olympus,Tokyo,Japan).The target cells were identified with an orientating beam and then captured by a laser beam.A total of 5000-10000 surface cuboidal cells and polygonal cells were captured.According PicoPure~(TM) RNA Isolation Kit(Arcturus Bioscience,Mountain View,CA,USA),we extracted total RNA and stored in -80℃.
4、RT-PCR
RT-PCR was performed using a RiboAmps HS RNA Amplification kit(Arcturus Bioscience) according to the manufacturer's instructions.The PCR products were separated by electrophoresis on a 1.5%agarose gel and stained with ethidium bromide. The target bands were analyzed densitometrically using a Gel Imaging System (BioImaging System,UVP,CA,USA).The b-actin gene was amplified as an internal control.
5.Laser capture microdissection of target cells,and extraction of DNA
The paraffin-embedded samples were sectioned successively at a thickness of 6 um.The sections were subjected to Mayer's H&E staining,dehydration with gradient alcohol,and lucidification with xylene(for 5 min).After drying,the sections were placed onto the object stage of an inverted microscope connected to a laser capture microdissection system (model LM200;Olympus,Tokyo,Japan).The target cells were identified with an orientating beam and then captured by a laser beam.A total of 5000-10000 surface cuboidal cells and polygonal cells were captured.A 0.5-ml centrifuge tube containing 50 ml DNA lysis buffer(10 mmol/1 Tris HC1,pH8.0;1 mmol/1 EDTA,pH 8.0;1%Tween 20; 200 mg/ml proteinase K) was incubated at 48℃for 14 h,proteinase K was deactivated at 95℃for 10 min,and the tube was stored 220℃until further analysis.
6、PCR-SSCP and Sequencing
PCR reaction mixture(50 ml) containing 10 ml DNA isolated from polygonal or surface cuboidal cells served as a template,and was mixed with 1 ml primers(30 pmol/ml;5-8 exons),0.4 ml Taq polymerase,4 ml dNTP,and 5 ml 106PCR buffer. The PCR conditions included initial denaturing at 94uC for 2 min,then 40 cycles at 94uC for 40 s,after which samples were subjected to annealing(see table 1 for temperatures and times),and a final extension at 72uC for 1 min.The PCR products(4 ml) were loaded onto a 2%agarose gel to confirm successful amplification and non-specific bands,followed by SSCP analysis.The PCR product(6 ml) was mixed with an equal volume of loading dye and denatured at 100uC for 10 min,and placed on ice for 5 min.The samples were then separated on 10%non-denaturing polyacrylamide gel(49:1).For each condition,we used adjacent normal lung tissue as a control.After electrophoresis,the gel was fixed,silver-stained,developed,photographed and analysed.The sample was considered normal if the band position was the same as that of the normal tissue.The same PCR products were used for sequencing(Shanghai United Gene Biotechnology,Shanghai,China).
Results
1、Gross and histological feature
The nodules were 1.4-4.9 cm in diameter and were well circumscribed with or without capsule.They were medium soft and often had a pale-brown region caused by haemorrhage.The tumour showed expansive growth pattern without multiple masses, infiltration and metastasis in any case.Histologically,all cases showed varying degrees of solid,papillary,haemorrhagic and sclerotic patterns.The tumours were composed entirely of polygonal and cuboidal cells.Polygonal cells were located in the solid and papillary areas,which had faintly stained or eosinophilic cytoplasm,round or oval nuclei,and small nucleoli,but rare or no karyomitosis.Surface cuboidal cells covered the papilla or were located in the interspaces of the haemorrhagic areas and in the lacuna spaces of the solid areas.These cells were mostly cuboidal,and some were thin and flat,or cylindrical.These cells had merged into multinucleated giant cells,3 but did not present as heteromorphic.Infiltration of inflammatory cells such as lymphocytes, infiltrated haemosiderin deposition,calcification,and ossification could be observed in the interstitium.
2、Immunohistochemistry
Epithelial markers were all positive in cuboidal cells of all patterns.In the solid regions, some cell clusters showed positive immunoreactivity with CK-L.The results of SP-B staining were identical to the results seen with epithelial markers in the cuboidal cells. The multinuclear giant cells derived from cuboidal cells were also positive to SP-B staining but negative to CD68.Some cuboidal cells remaining in the solid region of PSH also expressed the surfactant proteins.However,both epithelial markers and SP-B staining were negative in polygonal cells,which lined the stroma.Both cuboidal cells and polygonal cells in the nucleus were strongly positive for TTF-1.The results of electron microscopy revealed a few short microvilli on the cuboidal cell surface.The cuboidal cells also had abundant rough endoplasmic reticulum,mitochondria,and typical lamellar bodies for pneumocytes typeⅡ.However,no neuroendocrine granule was seen in the cuboidal cells.Some polygonal cells contained sparse neuroendocrine granules and abundant microfilaments besides abundant rough endoplasmic reticulum and mitochondria.On the contrary,no lamellar body was seen in these cells.In addition, 2 neuroendocrine markers,such as NSE,were positive or dispersed positive in the polygonal cells of every pattern.Other neuroendocrine marker was faintly positive in polygonal cells,including synaptophysin and chromogranin A.Vimentin staining was positive in all cases.CD34 expressed positive results only in the endothelial cells of small vessels and was negative in cuboidal cells and polygonal cells.Some positive cells for MCT were seen sparsely in papillary,solid,and sclerosing regions
p53 protein expression was observed in both cell types in three out of the 19 PSH tissue samples(15.8%),with more immunoreactive polygonal cells than immunoreactive surface cuboidal cells.Of the other 15 samples showing no p53 protein expression,one case exhibited immunoreactivity,but in fewer than 5%of cells.There was no p53 protein expression in any samples of normal lung tissue.
3、RT-PCR
Gene expressions of polygonal and cuboidal cells were significantly different. Surface cuboidal cell strongly expressed cytokeratin,epithelial membrane antigen, surfactant protein B,and thyroid transcription factor-1 mRNA,whereas interstitial polygonal cell strongly expressed vimentin,synaptophysin,and thyroid transcription factor-1 mRNA and weakly expressed epithelial membrane antigen.Neither cell type expressed chromogranin-A.
4、SSCP and sequencing analyses
The SSCP analysis and DNA sequencing revealed that abnormal mobility bands and mutations were observed in three p53-immunoreactive cases,with a mutation rate of 15.8%.A mutation in the p53 gene occurred in exon 6 in one case,and exon 7 in two cases.No mutations were found in exons 5 and 8.Two cases showed mutation only in the polygonal cells,while one case shows double(separate) mutations in both the polygonal and cuboidal cells.Four missense mutations were identified and their amino acid sequences were predicted.
Conclusions
1、The multinuclear giant cells in PSH are combined by the surface cuboidal cells. Multinuclear giant cells and sclerosing papillary are one of the reason of pathological misdiagnosis.
2、The differences in morphological phenotype might result from differences in the state of differentiation,cuboidal cells may differentiate into typeⅡpneumocytes, while polygonal in stroma possess the multipotency differentiation.
3、The expression of p53 protein may not be indicative of p53 gene mutation in PSH.The alteration of p53 gene and the expression of p53 protein are identified in both polygonal and cuboidal cells.The high mutation rate of p53 gene may indicate that PSH has potentially malignant biological behavior.
肺硬化性血管瘤(pulmonary sclerosing hemangioma)是由Liebow和Hulbell首先报道的肺部较为少见肿瘤,约占肺肿瘤的2-3%,最初认为是黄色瘤或错构瘤,长期以来被归类于肺的炎性假瘤范畴。随着时间的推移,人们逐渐认识到:肺的硬化性血管瘤并不是肺的炎性假瘤,而是一种实实在在的“良性”真性肿瘤,尽管其发病率较低,但由于临床CT、磁共振等影像学检查极易与肺癌相混淆,更为严重的是术中冰冻病理诊断又极易误诊为肺腺癌、细支气管肺泡癌以及类癌等,因而手术切除常常过度,给患者造成损害,所以对该肿瘤的研究和诊断备受重视。在我们的研究中,为了区分该肿瘤并不是炎性假瘤,也为了将来能够有一个更适合该肿瘤的名字。我们将该肿瘤称之为“所谓的肺硬化性血管瘤”(So-calledpulmonary sclerosing hemangioma,PSH)。由于该肿瘤一般呈良性临床经过,与其相平行的名称还有:良性乳头状神经内分泌肿瘤,良性肺泡细胞瘤,良性肺腺瘤,错构瘤等。之所以出现这些名称,是因为到目前为止PSH的起源尚不清楚。有人认为来源于内皮细胞、间皮细胞、肺泡上皮细胞、神经内分泌细胞等,最近还有人提出是来源于多潜能的原始呼吸道上皮细胞的观点。在新版的WHO分类当中,也只好将该肿瘤暂时放入“未定类肿瘤”当中。有人还对该肿瘤当中两种主要形态不同的细胞是否为同一起源提出了质疑,并观察到部分肿瘤术后若干年出现了复发和转移的现象。
针对PSH的分类困难、影像和病理诊断困难、起源不明、生物学特性不清的特点,我们收集了40余例的PSH资料,利用免疫组化、激光捕获微切割(LCM)、RT-PCR、单链构象多态性分析和DNA测序分析等方法,对该肿瘤进行系统深入的研究,期望找到能正确诊断该肿瘤的方法,并对其起源及生物学行为方面作以探讨。
材料与方法
1、材料
收集中国医科大学第一临床学院1995～2004年间病理诊断为PSH及手术切除病变旁正常肺组织蜡块标本40例。选其中17例女性,2例男性具有完整临床资料的病例进行基因表达方面的研究。该19例当中,12例无自觉症状,为体检时发现;7例有症状者分别表现为胸痛、咳嗽、痰中带血。胸部X线片或胸部CT显示均为孤立性肿块,多位于肺的周边,呈圆形、卵圆形高密度影,边缘光滑,无毛刺及分叶状改变。肿块位于右肺11例,左肺8例。未发现多发病灶及转移病例。发病时间为15天至5年不等。
2、免疫组化
选用单克隆抗体细胞角蛋白(cytokeratin L,CK-L)、上皮膜抗原(epithelialmembrane antigen,EMA)标记上皮性抗原,SP-B和TTF-1标记原始呼吸道上皮细胞或肺泡Ⅱ型细胞,波形蛋白(vimentin,VT)标记间叶来源细胞,CD34标记内皮来源细胞,CD68标记巨噬细胞,神经元特异性烯醇化酶(neuron specific endolase,NSE)和突触素(synaptophysin,Syn)标记神经内分泌来源的细胞以及单克隆P53抗体(DO-7)。上述即用型抗体及S-P超敏试剂盒均购自福州迈新物技术公司。将蜡块标本制成4μm连续切片,经脱蜡、脱苯、水化后进行HE和链菌素抗生物素蛋白-过氧化物酶(S-P法)免疫组化染色。以已知阳性组织为阳性对照,PBS缓冲液代替一抗为阴性对照。每张切片随机选取5个高倍视野,每高倍视野计数100个瘤细胞,无着色及阳性细胞数≤5%为(-),阳性细胞数＞5%为(+)。CK-L、SP-B、Vimentin、CD34、CD68、NSE、Syn阳性表达的判断为单纯胞质表达;EMA阳性表达的判断为膜/质表达;TTF-1、p53阳性表达的判断为单纯核表达或核、胞质同时表达。
3、激光捕获目的细胞、提取总RNA
取病变组织蜡块,制成6μm连续切片,Mayer's苏木素—伊红染色,二甲苯透明,切片晾干,放置在激光捕获显微切割(lasercapture microdissection,LCM)系统(LM200型)的倒置显微镜的载物台上。将附有一层乙烯乙酸乙烯酯薄膜(et hylenevinylacetate polymer,EVA)的扁平塑料帽紧密覆盖在切片的表面。定位光束标定被选细胞,发射激光束,分别捕获PSH组织中表面立方细胞和多角形细胞。每份标本分别捕获5000～10000个目的细胞。按照PicoPure~(TM) RNA Isolation Kit说明书操作提取总RNA(Arcturus Bioscience,Mt.View,CA)。
4、RT-PCR
按照RiboAmp(?)HS RNA Amplification Kit说明书操作。PCR扩增产物使用1.5%琼脂糖,100V电泳35min,溴化乙啶(EB)染色,凝胶成像系统(BioImageingSystem,UVP,CA)下观察。以β-actin为内对照。
5、DNA提取、P53基因PCR扩增、SSCP及测序分析
将捕获的PSH组织中表面立方细胞和多角形细胞加入预先加有50μL DNA裂解液(10 mmol/L Tris HCl,pH8.0;1 mmol/L EDTA PH8.0;1%Tween 20;200 mg/L蛋白酶K)的0.5 mL离心管上。48℃水浴14 h,95℃10 min灭活蛋白酶K,-20℃保存备用。分别取多角形细胞和立方细胞的DNA模板各10μL于50μLPCR反应体系进行P53基因(5～8外显子)PCR扩增。取6μL PCR产物加等量变性上样缓冲液混匀后,100℃热变性10 min后迅速置于冰水浴中冷却5 min,然后上样于10%非变性聚丙烯酰胺凝胶(49∶1)中;每组均以病变旁正常肺组织作对照。恒温电泳。凝胶经硝酸银染色。判断标准:待测样品的电泳带与正常对照的电泳带相比,若出现条带的增加、减少或位置的移动,即说明该样品存在着基因突变。同时取剩余的p53基因(5～8外显子)扩增产物(40μL左右)进行测序分析(上海联合基因测序部)。
结果
1、组织学改变
PSH组织形态多样,由实性区、乳头区、血管瘤样区和硬化区四种基本结构构成,并有不同程度的相互移行。肿瘤细胞主要有两种:一种是位于实性区和乳头区内的多角形瘤细胞,胞浆淡染或嗜酸,核圆或卵圆形,有小核仁,罕见或无核分裂像。另一种是被覆在乳头表面、血管瘤样区间隙及实性区裂隙内的细胞,多呈矮立方状,形态与肺泡Ⅱ型细胞极为相似,可融合成多核巨细胞,但无异型性。间质内可见较多的肥大细胞和以淋巴细胞为主的各种炎性细胞浸润,还可见含铁血黄素沉积、钙化和骨化等。多核瘤巨细胞和硬化性乳头的出现,是造成诊断困难的原因之一。
2、免疫组化结果
在40例PSH标本中,CK-L、EMA和SP-B只表达在表面立方上皮细胞或实性区内陷的立方上皮细胞和血管瘤样区的内衬上皮细胞,而间质多角形细胞为阴性;Vimentin则只表达于间质的多角形细胞;TTF-1在两种细胞内均有表达;CD34只在血管内皮细胞表达,在两种细胞内均无表达;多核瘤巨细胞则表达CK-L,SP-B和TTF-1,而CD68为阴性。此外,间质多角形细胞少量表达Syn和NSE。19例PSH组织中,3例(15.8%)标本p53蛋白呈阳性表达,且多角形细胞的表达强度高于表面立方细胞的表达;15例(78.9%)p53蛋白呈阴性表达,其中1例标本虽有p53蛋白呈阳性反应的细胞,但阳性细胞数≤5%。
3、RT-PCR结果
应用LCM方法,分别提取19例PSH当中的两种细胞的mRNA,RT-PCR扩增电泳后,所谓肺硬化性血管瘤组织中的两种细胞,其基因表达有着明显的区别,表面立方细胞有明确的CK、EMA、SP-B和TTF-1的mRNA表达,而间质内的多角形细胞有明确的Vimentin、Syn和TTF-1的表达,同时有弱的EMA的表达。两种细胞都未见Ch-A的表达。这一结果与免疫组化的蛋白表达结果极为相似。
4、SSCP及测序分析
SSCP法检测19例PSH组织P53基因突变,3例(15.8%)标本出现异常移动带,1例发生在第6外显子上,2例发生在第7外显子上,第5及第8外显子未见异常;在这3例突变中,单一多角形细胞突变2例,立方细胞和多角形细胞同时突变1例。DNA测序分析结果显示19例PSH组织中,P53基因的突变率为15.8%(3/19);单一多角形细胞突变2例,多角形细胞和立方细胞同时突变1例;1例发生在第6外显子;2例发生在第7外显子,第5、8外显子未见突变;3例均为错义突变。
结论
1、PSH组织当中的多核瘤巨细胞是表面立方细胞的融合,多核瘤巨细胞和硬化性乳头的出现,是造成病理诊断困难的主要原因之一。
2、PSH组织中的多角形细胞和表面立方细胞在形态表型上的差异可能是由于所处不同的分化状态所致,表面立方细胞趋向肺泡Ⅱ型细胞分化,而间质内的多角形细胞则具有多向分化的潜能。
3、PSH中两种细胞均可有突变型P53蛋白表达;多角形细胞中突变型p53蛋白表达率明显高于立方细胞;PSH中两种细胞均存在DNA错义突变,且间质多角形细胞突变高于表面立方细胞;P53蛋白表达与基因突变检测结果一致,提示PSH具有潜在恶性的可能。
Objective
Pulmonary sclerosing hemangioma(PSH) is an uncommon tumor of the lung and it s histogenesis and origin are uncertain to date.PSH consists of two major tumor cells: polyangular cells and cuboidal cells.Polyangular cells are true tumor cells which derive from primitive respiratory epithelium,but it remains controversial over whethere the surface lining cuboidal cells of PSH are true tumor cells or the results of responsive proliferation in the tumor genesis and whethere this two major tumor cells have the same origin.A general consensus appears to have been reached that PSH is a benign neoplasm,but a few PSHs are found to invade and metastasize.The phenomenon which lesion tissues invaded bronchus and peripheral mesenchyma can be seen reported.In order to further veritfy the origin and the possible character of PSH,we captured two major cells of PSH by laser capture micro-dissection(LCM).We selectedthe P53 gene which was related to pathogenesis of this disease as the subject of study and used immunohistochemical method,laser capture micro-dissection(LCM) technology,Single-stranded conformation polymorphism(SSCP) and DNA sequencing analysis to examine the expression of P53 protein and mutation of P53 gene in polyangular cells and cuboidal cells in PSH and valued its significance,providing reference to histogenesis and biological conduct of PSH.The expression of p53 protein and mutation of p53 gene are significant parameters which can reflect biological behavior of tumor cells.The aim of this study is to investigate the p53 protein expression and p53 gene mutation.Disclosing the different gene expression of the two cell types will provide the basis for the study of histogenesis of PSH tissue and helpful for differential diagnosis.
Materials and Methods
1、Patients
Paraffin-embedded PSH samples and normal lung tissue adjacent to PSH were obtained from 40 patients who were diagnosed with PSH at the First Clinical College of China Medical University between 1995 and 2004.Among those cases,19 cases were celected which had whole clinic data to do gene expression analysis.This study was conducted according to the regulations of the institutional review boards(China Medical University).The age range was 24-46 years,and the mean age was 34.5 years. Of the 19 patients,12 had no symptoms and their PSH was found upon routine examination;the other seven patients presented with symptoms including chest pain, cough and bloody phlegm.X Rays or CT scans revealed unitary masses,frequently in the periphery of the lungs.These masses were round or oval and of high density,with a smooth boundary,and with no burs or lobules.The masses were located in the right lung in 11 cases and the left lung in eight cases.The follow-up time(after surgery,to December 2005) ranged from 13-129 months;all patients survived,without recurrence or metastasis.
2、Immunohistochemistry
Low molecular weight cytokeratin(CK-L) and epithelial membrane antigen (EMA) sign epithelia antigen,surfactant protein B(SP-B) and thyroid transcription factor- 1(TTF-1) sign respiratory multipotential primitive epithelium or pneumocytes typeⅡ,Vimentin(VT) signs mesenchymal antigen,CD34 signs endothelial cells of vessels,CD68 signs macrophage,neuron specific endolase(NSE) and chromograninA(ChA) sign endocrine cell.Formalin-fixed paraffin-embedded tissue blocks were cut into 4 mm sections,de-waxed and hydrated.Immunostaining was performed with the streptavidin peroxidase system(Ultrasensitive;MaiXin,Fuzhou, China) according to manufacturer's instructions.The sections were incubated with a primary antibody(dilution 1:50;Santa Cruz Biotechnology,Santa Cruz,California, USA).Biotinylated goat anti-mouse serum IgG was used as a secondary antibody. After washing three times in phosphate-buffered saline(PBS),the sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase,and visualised by demonstration of conjugated peroxidase with diaminobenzidine as the substrate.The sections were counterstained with haematoxylin.For the negative control, primary antibodies were replaced with PBS;for the positive control,known positive tissue was used.A slide was considered negative or positive according to the absence or presence of positive staining:no staining or fewer than 5%of total cells positive for p53 was considered negative;greater than 5%of total cells positive for p53 was considered positive staining.
3、Laser capture microdissection of target cells,and extraction of RNA
The paraffin-embedded samples were sectioned successively at a thickness of 6 mm.The sections were subjected to Mayer's H&E staining,dehydration with gradient alcohol,and lucidification with xylene(for 5 min).After drying,the sections were placed onto the object stage of an inverted microscope connected to a laser capture microdissection system(model LM200;Olympus,Tokyo,Japan).The target cells were identified with an orientating beam and then captured by a laser beam.A total of 5000-10000 surface cuboidal cells and polygonal cells were captured.According PicoPure~(TM) RNA Isolation Kit(Arcturus Bioscience,Mountain View,CA,USA),we extracted total RNA and stored in -80℃.
4、RT-PCR
RT-PCR was performed using a RiboAmps HS RNA Amplification kit(Arcturus Bioscience) according to the manufacturer's instructions.The PCR products were separated by electrophoresis on a 1.5%agarose gel and stained with ethidium bromide. The target bands were analyzed densitometrically using a Gel Imaging System (BioImaging System,UVP,CA,USA).The b-actin gene was amplified as an internal control.
5.Laser capture microdissection of target cells,and extraction of DNA
The paraffin-embedded samples were sectioned successively at a thickness of 6 um.The sections were subjected to Mayer's H&E staining,dehydration with gradient alcohol,and lucidification with xylene(for 5 min).After drying,the sections were placed onto the object stage of an inverted microscope connected to a laser capture microdissection system (model LM200;Olympus,Tokyo,Japan).The target cells were identified with an orientating beam and then captured by a laser beam.A total of 5000-10000 surface cuboidal cells and polygonal cells were captured.A 0.5-ml centrifuge tube containing 50 ml DNA lysis buffer(10 mmol/1 Tris HC1,pH8.0;1 mmol/1 EDTA,pH 8.0;1%Tween 20; 200 mg/ml proteinase K) was incubated at 48℃for 14 h,proteinase K was deactivated at 95℃for 10 min,and the tube was stored 220℃until further analysis.
6、PCR-SSCP and Sequencing
PCR reaction mixture(50 ml) containing 10 ml DNA isolated from polygonal or surface cuboidal cells served as a template,and was mixed with 1 ml primers(30 pmol/ml;5-8 exons),0.4 ml Taq polymerase,4 ml dNTP,and 5 ml 106PCR buffer. The PCR conditions included initial denaturing at 94uC for 2 min,then 40 cycles at 94uC for 40 s,after which samples were subjected to annealing(see table 1 for temperatures and times),and a final extension at 72uC for 1 min.The PCR products(4 ml) were loaded onto a 2%agarose gel to confirm successful amplification and non-specific bands,followed by SSCP analysis.The PCR product(6 ml) was mixed with an equal volume of loading dye and denatured at 100uC for 10 min,and placed on ice for 5 min.The samples were then separated on 10%non-denaturing polyacrylamide gel(49:1).For each condition,we used adjacent normal lung tissue as a control.After electrophoresis,the gel was fixed,silver-stained,developed,photographed and analysed.The sample was considered normal if the band position was the same as that of the normal tissue.The same PCR products were used for sequencing(Shanghai United Gene Biotechnology,Shanghai,China).
Results
1、Gross and histological feature
The nodules were 1.4-4.9 cm in diameter and were well circumscribed with or without capsule.They were medium soft and often had a pale-brown region caused by haemorrhage.The tumour showed expansive growth pattern without multiple masses, infiltration and metastasis in any case.Histologically,all cases showed varying degrees of solid,papillary,haemorrhagic and sclerotic patterns.The tumours were composed entirely of polygonal and cuboidal cells.Polygonal cells were located in the solid and papillary areas,which had faintly stained or eosinophilic cytoplasm,round or oval nuclei,and small nucleoli,but rare or no karyomitosis.Surface cuboidal cells covered the papilla or were located in the interspaces of the haemorrhagic areas and in the lacuna spaces of the solid areas.These cells were mostly cuboidal,and some were thin and flat,or cylindrical.These cells had merged into multinucleated giant cells,3 but did not present as heteromorphic.Infiltration of inflammatory cells such as lymphocytes, infiltrated haemosiderin deposition,calcification,and ossification could be observed in the interstitium.
2、Immunohistochemistry
Epithelial markers were all positive in cuboidal cells of all patterns.In the solid regions, some cell clusters showed positive immunoreactivity with CK-L.The results of SP-B staining were identical to the results seen with epithelial markers in the cuboidal cells. The multinuclear giant cells derived from cuboidal cells were also positive to SP-B staining but negative to CD68.Some cuboidal cells remaining in the solid region of PSH also expressed the surfactant proteins.However,both epithelial markers and SP-B staining were negative in polygonal cells,which lined the stroma.Both cuboidal cells and polygonal cells in the nucleus were strongly positive for TTF-1.The results of electron microscopy revealed a few short microvilli on the cuboidal cell surface.The cuboidal cells also had abundant rough endoplasmic reticulum,mitochondria,and typical lamellar bodies for pneumocytes typeⅡ.However,no neuroendocrine granule was seen in the cuboidal cells.Some polygonal cells contained sparse neuroendocrine granules and abundant microfilaments besides abundant rough endoplasmic reticulum and mitochondria.On the contrary,no lamellar body was seen in these cells.In addition, 2 neuroendocrine markers,such as NSE,were positive or dispersed positive in the polygonal cells of every pattern.Other neuroendocrine marker was faintly positive in polygonal cells,including synaptophysin and chromogranin A.Vimentin staining was positive in all cases.CD34 expressed positive results only in the endothelial cells of small vessels and was negative in cuboidal cells and polygonal cells.Some positive cells for MCT were seen sparsely in papillary,solid,and sclerosing regions
p53 protein expression was observed in both cell types in three out of the 19 PSH tissue samples(15.8%),with more immunoreactive polygonal cells than immunoreactive surface cuboidal cells.Of the other 15 samples showing no p53 protein expression,one case exhibited immunoreactivity,but in fewer than 5%of cells.There was no p53 protein expression in any samples of normal lung tissue.
3、RT-PCR
Gene expressions of polygonal and cuboidal cells were significantly different. Surface cuboidal cell strongly expressed cytokeratin,epithelial membrane antigen, surfactant protein B,and thyroid transcription factor-1 mRNA,whereas interstitial polygonal cell strongly expressed vimentin,synaptophysin,and thyroid transcription factor-1 mRNA and weakly expressed epithelial membrane antigen.Neither cell type expressed chromogranin-A.
4、SSCP and sequencing analyses
The SSCP analysis and DNA sequencing revealed that abnormal mobility bands and mutations were observed in three p53-immunoreactive cases,with a mutation rate of 15.8%.A mutation in the p53 gene occurred in exon 6 in one case,and exon 7 in two cases.No mutations were found in exons 5 and 8.Two cases showed mutation only in the polygonal cells,while one case shows double(separate) mutations in both the polygonal and cuboidal cells.Four missense mutations were identified and their amino acid sequences were predicted.
Conclusions
1、The multinuclear giant cells in PSH are combined by the surface cuboidal cells. Multinuclear giant cells and sclerosing papillary are one of the reason of pathological misdiagnosis.
2、The differences in morphological phenotype might result from differences in the state of differentiation,cuboidal cells may differentiate into typeⅡpneumocytes, while polygonal in stroma possess the multipotency differentiation.
3、The expression of p53 protein may not be indicative of p53 gene mutation in PSH.The alteration of p53 gene and the expression of p53 protein are identified in both polygonal and cuboidal cells.The high mutation rate of p53 gene may indicate that PSH has potentially malignant biological behavior.
引文
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9 齐凤杰,张秀伟,张永兴,戴顺东,王恩华.所谓肺硬化性血管瘤组织中两种主要细胞的雄激素受体、磷酸甘油酸酯激酶基因多态性分析.中华病理学杂.2006,35(5):267-271.
10 Kazuto Yamazaki.Type-Ⅱ pneumocyte differentiation in pulmonary sclerosing hemangioma:ultrastructural differentiation and immunohistochemical distribution of lineage-specific transcription factors(TTF-1,HNF-3 alpha,and HNF-3 beta) and surfactant proteins.Virchows Arch (2004)445:45-53
11 Nakatani Y,Inayama Y,Kamijo S et al.Sclerosing lung hemagioma.Am J Surg Pathol,1999,23(2):240-243.
1 Devouassoux-Shisheboran M,Hayashi T,Linnoila RI,et al.A clinicopathologic study of 100 cases of pulmonary sclerosing hemangioma with immunohistochemical studies:TTF-1 is expressed in both round and surface cells,suggesting an origin from primitive respiratory epithelium.Am J Surg Pathol 2000;24:906-16.
2 Chan AC,Chan JK.Pulmonary sclerosing hemangioma consistently expresses thyroid transcription facter-1:a new clue to its histogenesis.Arch Pathol Lab Med 2001;125:1531-6.
3 Wang E,Lin D,Wang Y,et al.Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma.Hum Pathol 2004;35:503-8.
4 Niho S,Suzuki K,Yokose T,et al.Monoclonahty of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.Am J Pathol 1998;152:1065-9.
5 Dacic S,Sasatomi E,Swalsky PA,et al.Loss of heterozygosity patterns of sclerosing hemangioma of the lung and bronchioloalveolar carcinoma indicate a similar molecular pathogenesis.Arch Pathol Lab Med 2004;128:880-4.
6 Miyagawa-Hayashino A,Tazelaar HD,Langel DJ,et al.Pulmonary sclerosing hemangioma with lymph node metastases:report of 4 cases.Arch Pathol Lab Med 2003;127:321-5.
7 Shabnam MS,Srinivasan R,Wali A,et al.Expression of p53 protein and the apoptotic regulatory molecules Bcl-2,Bcl-XL,and Bax in locally advanced squamous cell carcinoma of the lung.Lung Cancer 2004;45:181-8.
8 Miller LD,Smeds J,George J,et al.An expression signature for p53 status in human breast cancer predicts mutation status,transcriptional effects,and patient survival.Proc Natl Acad Sci USA2005;102:3550-5.
9 Brambilla E,Negoescu A,Gazzeri S,et al.Apoptosis-related factors p53,Bc12,and Bax in neuroendocrine lung tumors.Am J Pathol 1996;149:1941-52.
10 Jackel MC,Sellmann L,Youssef S,et al.Prognostic significance of expression of p53,bcl-2 and bax in squamous epithelial carcinoma of the larynx—a multivariate analysis.HNO 2001;49:204-11.
11 Sirvent JJ,Aguilar MC,Olona M,et al.Prognostic value of apoptosis in breast cancer (pTl-pT2).A TUNEL,p53,bcl-2,bag-1 and Bax immunohistochemical study.Histol Histopathol 2004;19:759-70.
12 Bandoh N,Hayashi T,Kishibe K,et al.Prognostic value of p53 mutations,bax,and spontaneous apoptosis in maxillary sinus squamous cell carcinoma.Cancer 2002;94:1968-80.
13 Chiba W,Sawai S,Yasuda Y,et al.Positive expression of c-myc and p53 products in two cases of pulmonary sclerosing hemangioma.Nihon Kyobu Shikkan Gakkai Zasshi 1995;33:1348-54.
14 Iyoda A,Hiroshima K,Shiba M,et al.Clinicopathological analysis of pulmonary sclerosing hemangioma.Ann Thorac Surg 2004;78:1928-31.
15 Yano M,Yamakawa Y,Kiriyama M,et al.Sclerosing hemangioma with metastases to multiple nodal stations.Ann Thorac Surg 2002;73:981-3.
16 Jungraithmayr W,Eggeling S,Ludwig C,et al.Sclerosing hemangioma of the lung:a benign tumour with potential for malignancy? Ann Thorac Cardiovasc Surg 2006;12:352-4.
17 Chan NG,Melega DE,Inculet RI,et al.Pulmonary sclerosing hemangioma with lymph node metastases.Can Respir J 2003;10:391-2.
18 Katakura H,Sato M,Tanaka F,et al.Pulmonary sclerosing hemangioma with metastasis to the mediastinal lymph node.Ann Thorac Surg 2005;80:2351-3.
19 Komatsu T,Fukuse T,Wada H,et al.Pulmonary sclerosing hemangioma with pulmonary metastasis.Thorac Cardiovasc Surg 2006;54:348-9.
20 Kim KH,Sul HJ,Kang DY.Sclerosing hemangioma with lymph node metastasis.Yonsei Med J 2003;44:150-4.
21 Tanaka I,Inoue M,Matsui Y,et al.A case of pneumocytoma (so-called sclerosing hemangioma)with lymph node metastasis.Jpn J Clin Oncol 1986;16:77-86.
22 Nagata N,Dairaku M,Sueishi K,et al.Sclerosing hemangioma of the lung.An epithelial tumor composed of immunohistochemically heterogenous cells.Am J Clin Pathol 1987;8:552-9.
1 Liebow A A,Hubbell D S.Sclerosing hemangiom(histiocytoma,Xanthoma) of the lung.Cancer,1956,9:53-75.
2 Joshi K,Shankar SK,Gopinath N,et al.Multiple sclerosing haemangiomas of the lung.Postgrad Med J.1980,56(651):50-53.
3 Kim GY,Kim J,Choi YS,et al.Sixteen cases of sclerosing hemangioma of the lung including unusual presentations.J Korean Med Sci,2004,19(3):352-8.
4 Mojgan DS,Hayash i T,L inno ila R I,et al.A clinicopatho logic study of 100 cases of pulmonary sclero sing hemangioma with immunohis- tochemical studies.Am J Surg Pathol,2000,24(7):906-916.
5 Alvarez-Femandez E,Escalona-Zapata J.Sclerosing haemangioma of the lung.A histochemical,electron microscopical,tissue culture and time-lapse cinematographic study.Histopathology.1981,5(5):579-588.
6 Palacios J J N,Escribano P M,Toledo J,et al.Sclerosing hemangioma of the lung:an ultrastructural study.Cancer,1979,44:949-955.
7 Xu HM,Li WH,Hou N,e t al.Neuroendocrine differentiation in 32 cases of so2called sclerosing hemangioma of the lung:identified by immunohistochemical and ultrastructural study.Am J Surg Pathol,1997,21:1013-1022.
8 Saton Y,Tsuchiya E,Weng SY et al.Pulmonaty sclerosing hemangioma of the lung.A type Ⅱ pneumocytoma by immunohisto-chemical and immunoelectron microscopic studies.Cancer,1989,64(6):1310-1317.
9 Katzenstein AL,Fulling K,W eise DL,et al.So2called sclerosing hemangioma of the lung,evidence for mesothelial origin.Am J Surg Pathol,1983,7:3-14.
10 金锦善,段立疆,常占平等。肺硬化性血管瘤病理形态及免疫表型。临床与实验病理学杂志,2002,18(1):38-42。
11 张建强,周晓军,孟 奎,等.肺硬化性血管瘤组织中甲状腺转录因子-1的表达及其组织起源的探讨.中华病理学杂志,2002,31:42-45.
12 Devouassoux-Shisheboran M,Hayashi T,Linnoila RI,et al.A clinicopathologic study of 100cases of pulmonary sclerosing hemangioma with immunohistochemical studies:TTF-1 is expressed in both round and surface cells,suggesting an origin from primitive respiratory epithelium.Am J Surg Pathol,2006,24(7):906-916.
13 Kennedy A."Sclerosing hemangioma"of the lung:an alternative view of its development.J Clin Pathol,1973,26:792-799.
14 Yousem SA,Wick MR,Singh G,Katyal SL,So-called sclerosing hemangiomas of lung.An immunohistochemical study supporting a respiratory epithelial origin.Am J Surg Pathol.1988,12(8):582-590.
15 Stahlman MT,Gray ME,Whitsett JA.Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung.J Histochem Cytochem,1996,44:673-678.
16 Khoor A,Whitsett J A,Stahlman M T,et al.Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenoearcinoma of the lung from malignant mesothelioma.Hum Pathol,1999,30:695-700.
17 Khoor A,Gray ME,Hull WM,et al.Stahlman MT.Dvelopmental expression of SP-A mRNA in the proximal and distal respiratioty epithelium in the humen fetus and newborn.J Histoehem Cytochem 1993,41,1311-1319.
18 Lazzaro D,Price M,de Feliee M,et al.The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the fetal brain.Development,1991,113:1093-1104.
19 孔洁等。临床肺科杂志2007,8(5):456-458
20 王妍,王恩华,吴广平等。肺硬化性血管瘤免疫标记及超微结构-提示其细胞的不同来源。中国肺癌杂志,2003,4(6):92-96。
21 Anna-Luise A,Katzenstein,M,D.John J,et al.Sclerosing hemangioma of the lung.Am J Surg Pathol,1980,4,343-356.
22 Nagata N,Dairaku M,Sueishi K et al.Sclerosing hemangioma of the lung.An epithelial tumor composed of immunohistochemically heterogenous cells.A m J Clin Pathol,1987,88(5):552-559.
23 Niho S,Suzuki K,Yokose T,et al.Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.Am J Pathol,2006,52:1065-1069.
24 Tanaka I,Inoue M,Matsui Y,et al.A case of pneumocytoma (so-called sclerosing hemangioma)with lymph node metastasis.Jpn J Clin Oncol,1986,16(1):77-86.
25 Chan AC,Chan JK.Pulmonary sclerosing hemangioma consistently expresses thyroid transcription factor-l(TTF-l):a new clue to its histogenesis.Am J Surg Pathol,2000,24(11):1531-1536.
26 Miyagawa-Hayashino A,Tazelaar HD,Langel DJ,et al.Pulmonary sclerosing hemangioma with lymph node metastases:report of 4 cases.Arch Pathol Lab Med,2007,127(3):321-5.
27 Spencer H,Nambu S.Sclerosing haemangiomas of the lung.Histopathology.1986 May;10(5):477-87.
2 Hill GS,Eggleston JC.Electron microscopic study of so-called "pulmonary sclerosing hemangioma".Report of a case suggesting epithelial origin.Cancer.1972,30(4):1092-1096.
3 Wang E,Lin D,Wang Y,et al.Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma.Hum Pathol,2004,35(4):503-508.
4 Xu HM,Li WH,Hou N,Zhang SG,Li HF,Wang SQ,Yu ZY,Li ZJ,Zeng MY,Zhu GM,Neuroendocrine differentiation in 32 cases of so-called sclerosing hemangioma of the lung:identified by immunohistochemical and ultrastructural study.Am J Surg Pathol.1997 Sep;21(9):1013-22.
5 Kay S,Still WJ,Borochovitz D,Sclerosing hemangioma of the lung:an endothelial or epithelial neoplasm? Hum Pathol.1977 Jul;8(4):468-74.
6 Katzenstein AL,Weise DL,Fulling K,Battifora H.Neuroendocrine differentiation in 32 cases of so-called sclerosing hemangioma of the lung:identified by immunohistoehemical and ultrastructural study.Am J Surg Pathol,1983 Jan;7(1):3-14.
7 Niho S,Suzuki K,Yokose T,et al.Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.Am J Pathol,1998,152(4):1065-1069.
8 Nagata N,Dairaku M,Sueishi K,Tanaka K,et al.Sclerosing hemangioma of the lung.An epithelial tumor composed of immunohistochemically heterogenous cells,Am J Clin Pathol.1987 Nov;88(5):552-9
9 齐凤杰,张秀伟,张永兴,戴顺东,王恩华.所谓肺硬化性血管瘤组织中两种主要细胞的雄激素受体、磷酸甘油酸酯激酶基因多态性分析.中华病理学杂.2006,35(5):267-271.
10 Kazuto Yamazaki.Type-Ⅱ pneumocyte differentiation in pulmonary sclerosing hemangioma:ultrastructural differentiation and immunohistochemical distribution of lineage-specific transcription factors(TTF-1,HNF-3 alpha,and HNF-3 beta) and surfactant proteins.Virchows Arch (2004)445:45-53
11 Nakatani Y,Inayama Y,Kamijo S et al.Sclerosing lung hemagioma.Am J Surg Pathol,1999,23(2):240-243.
1 Devouassoux-Shisheboran M,Hayashi T,Linnoila RI,et al.A clinicopathologic study of 100 cases of pulmonary sclerosing hemangioma with immunohistochemical studies:TTF-1 is expressed in both round and surface cells,suggesting an origin from primitive respiratory epithelium.Am J Surg Pathol 2000;24:906-16.
2 Chan AC,Chan JK.Pulmonary sclerosing hemangioma consistently expresses thyroid transcription facter-1:a new clue to its histogenesis.Arch Pathol Lab Med 2001;125:1531-6.
3 Wang E,Lin D,Wang Y,et al.Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma.Hum Pathol 2004;35:503-8.
4 Niho S,Suzuki K,Yokose T,et al.Monoclonahty of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.Am J Pathol 1998;152:1065-9.
5 Dacic S,Sasatomi E,Swalsky PA,et al.Loss of heterozygosity patterns of sclerosing hemangioma of the lung and bronchioloalveolar carcinoma indicate a similar molecular pathogenesis.Arch Pathol Lab Med 2004;128:880-4.
6 Miyagawa-Hayashino A,Tazelaar HD,Langel DJ,et al.Pulmonary sclerosing hemangioma with lymph node metastases:report of 4 cases.Arch Pathol Lab Med 2003;127:321-5.
7 Shabnam MS,Srinivasan R,Wali A,et al.Expression of p53 protein and the apoptotic regulatory molecules Bcl-2,Bcl-XL,and Bax in locally advanced squamous cell carcinoma of the lung.Lung Cancer 2004;45:181-8.
8 Miller LD,Smeds J,George J,et al.An expression signature for p53 status in human breast cancer predicts mutation status,transcriptional effects,and patient survival.Proc Natl Acad Sci USA2005;102:3550-5.
9 Brambilla E,Negoescu A,Gazzeri S,et al.Apoptosis-related factors p53,Bc12,and Bax in neuroendocrine lung tumors.Am J Pathol 1996;149:1941-52.
10 Jackel MC,Sellmann L,Youssef S,et al.Prognostic significance of expression of p53,bcl-2 and bax in squamous epithelial carcinoma of the larynx—a multivariate analysis.HNO 2001;49:204-11.
11 Sirvent JJ,Aguilar MC,Olona M,et al.Prognostic value of apoptosis in breast cancer (pTl-pT2).A TUNEL,p53,bcl-2,bag-1 and Bax immunohistochemical study.Histol Histopathol 2004;19:759-70.
12 Bandoh N,Hayashi T,Kishibe K,et al.Prognostic value of p53 mutations,bax,and spontaneous apoptosis in maxillary sinus squamous cell carcinoma.Cancer 2002;94:1968-80.
13 Chiba W,Sawai S,Yasuda Y,et al.Positive expression of c-myc and p53 products in two cases of pulmonary sclerosing hemangioma.Nihon Kyobu Shikkan Gakkai Zasshi 1995;33:1348-54.
14 Iyoda A,Hiroshima K,Shiba M,et al.Clinicopathological analysis of pulmonary sclerosing hemangioma.Ann Thorac Surg 2004;78:1928-31.
15 Yano M,Yamakawa Y,Kiriyama M,et al.Sclerosing hemangioma with metastases to multiple nodal stations.Ann Thorac Surg 2002;73:981-3.
16 Jungraithmayr W,Eggeling S,Ludwig C,et al.Sclerosing hemangioma of the lung:a benign tumour with potential for malignancy? Ann Thorac Cardiovasc Surg 2006;12:352-4.
17 Chan NG,Melega DE,Inculet RI,et al.Pulmonary sclerosing hemangioma with lymph node metastases.Can Respir J 2003;10:391-2.
18 Katakura H,Sato M,Tanaka F,et al.Pulmonary sclerosing hemangioma with metastasis to the mediastinal lymph node.Ann Thorac Surg 2005;80:2351-3.
19 Komatsu T,Fukuse T,Wada H,et al.Pulmonary sclerosing hemangioma with pulmonary metastasis.Thorac Cardiovasc Surg 2006;54:348-9.
20 Kim KH,Sul HJ,Kang DY.Sclerosing hemangioma with lymph node metastasis.Yonsei Med J 2003;44:150-4.
21 Tanaka I,Inoue M,Matsui Y,et al.A case of pneumocytoma (so-called sclerosing hemangioma)with lymph node metastasis.Jpn J Clin Oncol 1986;16:77-86.
22 Nagata N,Dairaku M,Sueishi K,et al.Sclerosing hemangioma of the lung.An epithelial tumor composed of immunohistochemically heterogenous cells.Am J Clin Pathol 1987;8:552-9.
1 Liebow A A,Hubbell D S.Sclerosing hemangiom(histiocytoma,Xanthoma) of the lung.Cancer,1956,9:53-75.
2 Joshi K,Shankar SK,Gopinath N,et al.Multiple sclerosing haemangiomas of the lung.Postgrad Med J.1980,56(651):50-53.
3 Kim GY,Kim J,Choi YS,et al.Sixteen cases of sclerosing hemangioma of the lung including unusual presentations.J Korean Med Sci,2004,19(3):352-8.
4 Mojgan DS,Hayash i T,L inno ila R I,et al.A clinicopatho logic study of 100 cases of pulmonary sclero sing hemangioma with immunohis- tochemical studies.Am J Surg Pathol,2000,24(7):906-916.
5 Alvarez-Femandez E,Escalona-Zapata J.Sclerosing haemangioma of the lung.A histochemical,electron microscopical,tissue culture and time-lapse cinematographic study.Histopathology.1981,5(5):579-588.
6 Palacios J J N,Escribano P M,Toledo J,et al.Sclerosing hemangioma of the lung:an ultrastructural study.Cancer,1979,44:949-955.
7 Xu HM,Li WH,Hou N,e t al.Neuroendocrine differentiation in 32 cases of so2called sclerosing hemangioma of the lung:identified by immunohistochemical and ultrastructural study.Am J Surg Pathol,1997,21:1013-1022.
8 Saton Y,Tsuchiya E,Weng SY et al.Pulmonaty sclerosing hemangioma of the lung.A type Ⅱ pneumocytoma by immunohisto-chemical and immunoelectron microscopic studies.Cancer,1989,64(6):1310-1317.
9 Katzenstein AL,Fulling K,W eise DL,et al.So2called sclerosing hemangioma of the lung,evidence for mesothelial origin.Am J Surg Pathol,1983,7:3-14.
10 金锦善,段立疆,常占平等。肺硬化性血管瘤病理形态及免疫表型。临床与实验病理学杂志,2002,18(1):38-42。
11 张建强,周晓军,孟 奎,等.肺硬化性血管瘤组织中甲状腺转录因子-1的表达及其组织起源的探讨.中华病理学杂志,2002,31:42-45.
12 Devouassoux-Shisheboran M,Hayashi T,Linnoila RI,et al.A clinicopathologic study of 100cases of pulmonary sclerosing hemangioma with immunohistochemical studies:TTF-1 is expressed in both round and surface cells,suggesting an origin from primitive respiratory epithelium.Am J Surg Pathol,2006,24(7):906-916.
13 Kennedy A."Sclerosing hemangioma"of the lung:an alternative view of its development.J Clin Pathol,1973,26:792-799.
14 Yousem SA,Wick MR,Singh G,Katyal SL,So-called sclerosing hemangiomas of lung.An immunohistochemical study supporting a respiratory epithelial origin.Am J Surg Pathol.1988,12(8):582-590.
15 Stahlman MT,Gray ME,Whitsett JA.Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung.J Histochem Cytochem,1996,44:673-678.
16 Khoor A,Whitsett J A,Stahlman M T,et al.Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenoearcinoma of the lung from malignant mesothelioma.Hum Pathol,1999,30:695-700.
17 Khoor A,Gray ME,Hull WM,et al.Stahlman MT.Dvelopmental expression of SP-A mRNA in the proximal and distal respiratioty epithelium in the humen fetus and newborn.J Histoehem Cytochem 1993,41,1311-1319.
18 Lazzaro D,Price M,de Feliee M,et al.The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the fetal brain.Development,1991,113:1093-1104.
19 孔洁等。临床肺科杂志2007,8(5):456-458
20 王妍,王恩华,吴广平等。肺硬化性血管瘤免疫标记及超微结构-提示其细胞的不同来源。中国肺癌杂志,2003,4(6):92-96。
21 Anna-Luise A,Katzenstein,M,D.John J,et al.Sclerosing hemangioma of the lung.Am J Surg Pathol,1980,4,343-356.
22 Nagata N,Dairaku M,Sueishi K et al.Sclerosing hemangioma of the lung.An epithelial tumor composed of immunohistochemically heterogenous cells.A m J Clin Pathol,1987,88(5):552-559.
23 Niho S,Suzuki K,Yokose T,et al.Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung.Am J Pathol,2006,52:1065-1069.
24 Tanaka I,Inoue M,Matsui Y,et al.A case of pneumocytoma (so-called sclerosing hemangioma)with lymph node metastasis.Jpn J Clin Oncol,1986,16(1):77-86.
25 Chan AC,Chan JK.Pulmonary sclerosing hemangioma consistently expresses thyroid transcription factor-l(TTF-l):a new clue to its histogenesis.Am J Surg Pathol,2000,24(11):1531-1536.
26 Miyagawa-Hayashino A,Tazelaar HD,Langel DJ,et al.Pulmonary sclerosing hemangioma with lymph node metastases:report of 4 cases.Arch Pathol Lab Med,2007,127(3):321-5.
27 Spencer H,Nambu S.Sclerosing haemangiomas of the lung.Histopathology.1986 May;10(5):477-87.