摘要
目的
过氧化物酶体增殖物激活型受体γ(PPARγ)是近来新发现的核内激素类受体家族成员，是配体激活的转录因子，它能够结合位于靶基因增强子中的过氧化物酶体增殖反应元件，调节靶基因的转录，有研究证实PPARγ在细胞分化、肿瘤侵袭、转移中发挥重要作用，但在妊娠滋养细胞疾病(GTD)中研究较少。本研究拟探讨在GTD中PPARγ蛋白的表达及其意义。
实验材料方法
一、材料
收集1988-2006年中国医科大学附属第二医院妇产科和丹东市妇女儿童医院妇产科手术切除的葡萄胎组60例，平均年龄(27.14±8.03岁)，均为首次清宫标本(随访两年未发生恶变)。根据滋养细胞增生程度分为3度：轻度26例、中度20例、重度14例。侵蚀性葡萄胎组12例，平均年龄(30.23±8.87岁)，临床分期Ⅰ期9例，Ⅱ期及其以上3例。绒毛膜上皮癌组11例，平均年龄(34.46±9.44岁)，临床分期Ⅰ期2例，Ⅱ期及其以上9例。另有正常妊娠组30例(12-14孕周)，胎盘绒毛组织均取自2005年6月至2006年1月在中国医科大学第二附属医院计划生育门诊就诊的正常孕妇，平均年龄(26.97±5.13岁)，均未服用紧急避孕药，无合并症和并发症，均已签知情同意书。
二、方法
所有组织均经4％的多聚甲醛固定，常规石蜡包埋，4μm连续切片，每张切片经常规脱蜡、水化，3％过氧化氢去除内源性过氧化酶，切片需经微波抗原修复，滴入非免疫性动物血清封闭。滴加PPARγ多克隆抗体约1滴，4℃过夜，冲洗后切片再加1滴生物素标记的第二抗体，室温孵育30分钟后，PBS冲洗，DAB显色，苏木素复染色，常规脱水，透明树胶封固。用PBS代替一抗作阴性对照，用已知阳性标本作阳性对照。
三、统计学分析
计数资料采用SPSS10.0统计软件进行x~2检验。
实验结果
1、PPARγ蛋白阳性表达主要定位于细胞核中。PPARγ蛋白阳性表达在正常妊娠胎盘绒毛主要定位于细胞滋养细胞核中，并在细胞柱和细胞岛中的细胞滋养细胞核中呈高表达。在GTD中PPARγ蛋白虽也定位于细胞滋养细胞核中，但阳性表达变化较大，在葡萄胎组织中多为阳性表达，虽分布不规律，但与正常妊娠绒毛比较无差异(P＞0.05)。在侵蚀性葡萄胎组织中表达下降，显著低于正常早孕绒毛和葡萄胎组织(P＜0.01)。在绒毛膜上皮癌组织中阳性表达明显下降，与正常妊娠绒毛组织和葡萄胎组织比较差异显著(P＜0.01)。PPARγ在绒毛膜上皮癌中的阳性表达率低于侵蚀性葡萄胎，但无统计学差异(P＞0.05)。
2、PPARγ蛋白在滋养细胞轻度增生的葡萄胎组织中多数为强阳性表达，中度增生中表达略有下降，但与前者比较无差异(P＞0.05)，在重度增生中表达明显下降，与轻度增生比较有差异(P＜0.05)，与中度增生比较无差异(P＞0.05)。
结论
1、PPARγ蛋白主要定位于细胞核中，PPARγ蛋白在正常绒毛和葡萄胎组织中为高表达，在绒毛膜上皮癌组织中表达明显减少或不表达，而在侵蚀性葡萄胎组织中PPARγ蛋白表达介于二者之间。
2、PPARγ蛋白在滋养细胞增生程度不同的葡萄胎组织中表达有差异(P＜0.05)，即滋养细胞增生越明显，PPARγ蛋白表达越弱。
Objective
Peroxisome proliferation activated receptory(PPARγ) belongs to nucleus hormone receptor superfamily new member. PPARγis an ligand activated transcription factor,it binds with specific DNA response element, then controls gene express. Maim function of PPARγhas been proved that it plays an important role of the messenger in the regulation of the cell differentiation、the cell proliferation the tumour invasion and metastasis. It is short comparatively in studying in gestational trophoblastic disease. This study was conducted to investigate the expression of PPARγprotein in gestational trophoblastic disease as well as to elucidate its significance.
Materials and Methods
1. Materials
Paraffin-embedded tissues from 60 patients with hydatidiform mole、12 patients with malignant mole、11 patients with trophoblastoma and from 30 case of normal placenta tissues from patients who received artificial abortion at the affiliated hospital of China Medical University between 1988-2006 were analyzed for PPARγprotein expression by immunohistochemical technique, patients with hydatidiform mole with a mean age of 27(27.14±8.03). patients with malignant mole with a mean age of 30 (30.23±8.87). patients with trophoblastoma with a mean age of 34 (34.46±9.44). patients with artificial abortion with a mean age of 26 (26.97±5.13).They had not received either chemotherapy or radiation therapy before surgery and agreed with this experiment.
2. Methods
All specimens were fixed in 4% buffered formaldehyle and prossed into routinely paraffin-embedded blocks. For each case, four-micrometer sections from these blocks were mounted on silane-coated slides, air-dried. Sections were dewatered and hydrated, and then treated in 3% hydrogen peroxide for 10 min to quench endoengous peroxidase. Prior to staining with the anti-PPARγantibodies, microwave antigen-retrienval technology was used, and then non-immuno animal serum was droped to seal. All procedures were performed at room temperature and in a moist chamker, with a PBS wash after each step. PPARγantibody were droped on the slides. After over night at 4℃, the sections were incubated with them, then after the flushing the slice again, the sections were added a drop of living creature second antibody, after the room temperature fosters 30 minutes, PBS flushes, the DAB coloration, the haematoxylin counterstain, the conventional dehydration, the transparent gum seals solidly.Replaces with PBS first anti-makes the negative comparison, makes the positive comparison with the known positive specimen.
3. Statistical methods
Statistical analyses of PPARγprotein expression was carried out by SSPS10.0. Differences were considered significant when P values were less than 0.05.
Results
1. The expression of PPARγprotein in the four kind of tissue was mostly located at cell nuclei.In normal first trimester tissues, PPARγprotein showed high expression, it was mainiy localized in the nuclei of the villous cytotrophoblast cells, Extravillous cytotrophoblast of cell island and cell columns also showed nuclear PPARγimmunostaining.A striking result was the altered expression patterns of PPARγin pathological tissues.In hydatidiform mole, it was found that It's positive expression, PPARγwas mainly localized in the nuclei of the trophoblastic collections of the pathological villi and in the extravillous trophoblastic cells, the positive cell distributed dispersively or locally. In malignant moles,PPARγshowed a reduced immunostaining, whereas in the choriocarcinoma,only a few trophoblastic cells showed weak PPARγnuclear immunostaining or even without staining.
2. The positive expression of PPARγprotein in hydatidiform moles is 78.57% in high degree hyperplasia, it was 100% in low degree hyperplasia. There is significantly difference between them(P＜0.05). The expression of PPARγprotein in moderate degree hyperplasia of hydatidiform moles is between in high degree hyperplasia and in low degree hyperplasia.
Conclusion
1. The expression of PPARγprotein was mostly located in the cell nuclei, The PPARγprotein In normal first trimester tissues and In hydatidiform moles showed high expression, but the expression of PPARγprotein decreased or loss significantly in the choriocarcinoma. The expression of PPARγis between the hydatidiform moles and the choriocarcinoma in malignant moles.
2. The expression of PPARγprotein is distinguish among different hyperplasia degree of trophocyte cells (P＜0.05),the higher was the hyperplasia of hydatidiform moles,the weaker would be the expression of PPARγprotein.
引文
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