摘要
研究背景
结直肠癌是威胁人类健康的主要疾病之一,我国结直肠癌的发病率有不断上升的趋势。传统的治疗方法有其自身的局限性,因此寻找新的疗法迫在眉睫。随着人们对大肠癌认识的加深及医学分子生物学研究的发展,肿瘤的基因治疗成为一种新的治疗模式。
越来越多的资料显示与细胞恶性增殖和分化受阻一样,细胞凋亡异常在大多数恶性肿瘤的发病学上亦占有重要地位。凋亡在正常情况下可以消灭DNA受损或细胞循环周期失调的细胞。肿瘤细胞的增殖率并不总是高于相应的正常细胞,这表明肿瘤细胞的积累是细胞逃过“凋亡排除”作用的结果。凋亡调控失调可以异常地延长细胞的生存时间,促进有转化作用的基因突变的积累,从而参与肿瘤的发生。所以肿瘤不仅是细胞增殖分化异常疾病,也是凋亡异常疾病。
诱导凋亡是一种新的肿瘤治疗方法,在肿瘤细胞内导入凋亡活化基因或灭活凋亡抑制基因是肿瘤基因治疗中最诱人的策略之一。Survivin是迄今发现的最强的凋亡抑制因子,survivin在癌组织中的表达与恶性肿瘤的侵袭性、预后均有一定关系。作为一个新的凋亡抑制蛋白,survivin在凋亡调节中起着重要作用。与其它的IAP家族成员不同,survivin在人胚胎组织中高度表达并参与胚胎发育过程中的组织分化和器官形成,在终末分化的成人组织中未有检出,但广泛表达于多种肿瘤组织。体内外研究表明,在许多恶性实体瘤如肺癌、结直肠癌、胰腺癌、前列腺癌和胃肠癌以及一些造血系统恶性肿瘤如非何杰金淋巴瘤中均有很强的表达。相反,在肿瘤周围的正常组织中未见表达。因此,survivin可作为一个肿瘤细胞治疗的理想靶点。
反义寡核苷酸(antisense oligonucleotides,ASODN)通过序列特异性地与靶mRNA结合而阻断mRNA翻译,调节由基因到蛋白质的信息传递,抑制蛋白质的表达。ASODN是根据中心法则从DNA水平入手,作用于遗传信息的上游,所需剂量较小,因此其药效高。由于ASODN'能特异性地阻断细胞内单一基因的表达,而不影响其他基因的正常功能,所以特异性强,副作用小。由于survivin只在胚胎和恶性肿瘤组织中表达,反义survivin基因治疗具有良好的靶向性、特异性及安全性。
基因治疗的重要目标是获得遗传物质在转导细胞中的持久整合及稳定表达,由于ASODN是亲水性的多聚阴离子聚合物,对生物膜的通透性差,具有不易透过细胞膜、细胞难于吸收、胞内积累少以及易受血浆和细胞内的核酸酶降解的特点,限制了它的广泛应用。为了增加细胞对ASODN的摄入率和稳定性,而不降低转移性,需要使用高效载体传递ASODN。
传统的病毒和非病毒载体在转染效率和安全性方面都存在各种不足,自纳米技术产生以后,用纳米颗粒作为基因转移载体,已引起医学界广泛重视。纳米微粒是一类由天然或合成的高分子物质组成的超微固态胶状粒子,粒径多在10-100nm范围内,大小与病毒相仿,比一般细胞小,可将DNA、RNA、寡核苷酸等生物活性分子包裹在其中或由静电相互吸引或吸附在其表面形成复合物,细胞摄取纳米微粒后,通过一系列复杂的过程释放出这些活性分子,从而发挥基因的治疗作用。聚酰胺-胺型树状大分子(polyamidoamine,PAMAM)是近年来国外开发的一类新型功能高分子,其分子在结构上具有高度的几何对称性、精确的分子结构、大量的官能团、分子内存在空腔及分子链增长具有可控性。多种细胞系中研究显示,以PAMAM为载体均较其它阳离子载体(如多聚赖氨酸、聚氮丙啶、脂质体)介导的基因转移率显著提高。寡核苷酸/PAMAM复合物不仅促进细胞对DNA的摄取,同时还可延长DNA在细胞内的存留时间。PAMAM具有保护DNA不被血清或组织中各种补体及酶所降解的作用,当DNA与PAMAM结合后,可抵御酶的降解。
基于上述研究,为提高survivin反义寡核苷酸的转染效率,增强基因治疗的效果,本课题拟应用PAMAM作为survivin-asODN的载体,配制成纳米转染复合物,检测其表征、基因载药率和体外DNA释放特征。并观察survivin-asODN对结直肠癌SW620细胞的体外杀伤作用及其体内抑瘤效应,为进一步开展结直肠癌的基因治疗研究提供实验依据。
目的
研究PAMAM作为survivin-asODN转染载体的可行性。
观察survivin反义寡核苷酸对SW620细胞中survivin基因表达的封闭作用和细胞生长的抑制作用。
最后,以SW620细胞株建立裸鼠皮下移植瘤动物模型,观察经PAMAM转导的survivin-asODN在裸鼠体内的抗肿瘤作用。
方法
一、PAMAM-survivin-asODN转染复合物的合成、形态观察、粒径与zeta电位测定、核酸载药率与包封率测定、体外基因释放测定以及琼脂糖凝胶电泳阻滞分析和抗核酸酶试验。
针对survivin mRNA翻译起始位点设计并合成反义寡核苷酸,与PAMAM按一定比例混合成PAMAM-survivin-asODN转染复合物,透射电子显微镜下观察粒子的形态,激光散射粒径仪测定转染复合物的粒径,zeta电位分析仪测定转染复合物的zeta电位,紫外分光光度计测定核酸载药率与包封率以及体外基因释放,PAMAM-survivin-asODN转染复合物电泳阻滞分析,加入核酸酶后电泳阻滞分析。
二、PAMAM介导survivin反义寡核苷酸(antisense oligonucleotide,ASODN)转染SW620细胞以及诱导细胞凋亡。
Survivin反义寡核苷酸通过PAMAM介导转染SW620细胞,采用westemblot、RT-PCR方法检测survivin反义寡核苷酸对SW620细胞survivin蛋白及mRNA表达的作用。测定转染前后细胞增殖活性变化,流式细胞仪检测细胞基因转染效率和凋亡率的变化。
三、PAMAM介导的survivin反义寡核苷酸对结直肠癌SW620细胞裸鼠模型的体内抑瘤效应。
采用BALB/C雌性裸鼠,4～6周龄,对数生长期的SW620细胞接种于裸鼠腹部皮下,建立结直肠癌动物模型,当肿瘤达0.5cm直径时,将荷瘤裸鼠随机分为3组,A组:阴性对照组(瘤体及瘤周注射Lipolifectmain~(TM2000)与PAMAM);B组:脂质体-survivin-asODN治疗组(瘤体及瘤周注射脂质体-survivin-asODN转染复合物);C组:PAMAM-survivin-asODN治疗组(瘤体及瘤周注射PAMAM-survivin-asODN转染复合物)。治疗前后测量肿瘤体积,治疗结束后测肿瘤重量,计算肿瘤抑瘤率,观察该治疗体系的体内抑瘤效应。治疗结束后行组织内survivin基因表达的检测、肿瘤组织透射电镜观察肿瘤细胞的超微结构变化及心、肝、脾、肺、肾组织病理检查。
结果
一、转染复合物的形态观察、粒径与zeta电位测定
透射电镜下观察脂质体-survivin-asODN和PAMAM-survivin-asODN复合物呈圆形或类圆形颗粒,脂质体-survivin-asODN复合物的粒径大于PAMAM-survivin-asODN复合物的粒径,差异有统计学意义(t=7.874,P=0.000,P＜0.001),而其zeta电位低于PAMAM-survivin-asODN复合物,差异有统计学意义(t=4.158,P=0.002,P＜0.01)。
二、转染复合物的核酸载药率,包封率及体外基因释放测定
脂质体-survivin-asODN和PAMAM-survivin-asODN复合物之间包封率差异无统计学意义(t=0.178,P=0.863,P＞0.05),载药率差异无统计学意义(t=0.582,P=0.573,P＞0.05)。脂质体-survivin-asODN复合物24～48 h内有爆破释放,然后迅速下降,6 d时已检测不到DNA释放;PAMAM-survivin-asODN复合物24～48h内有爆破释放,然后缓慢下降,平稳持续14 d以上,表明PAMAM对DNA具有缓释现象。
三、细胞转染及其转染效率
转染24 h后,荧光显微镜下观察两组细胞内均可见绿色荧光。48 h可获得最高转染效率,脂质体组细胞内的荧光强度(46.46±9.84)低于PAMAM组细胞(63.90±10.29),两者之间差异有统计学意义(t=2.739,P=0.025,P＜0.05)。
四、survivin反义寡核苷酸对SW620细胞的生长抑制效应
MTT法检测各组细胞生长抑制率:survivin-asODN作用的两组与空白对照组相比,细胞生长抑制率差异有统计学意义,经survivin-asODN作用后的细胞生长受到抑制(P＜0.001)。经PAMAM-survivin-asODN复合物和脂质体-survivin-asODN复合物处理的细胞均受到抑制,PAMAM-survivin-asODN复合物的作用更强,两者之间差异有统计学意义(P＜0.05)。
五、SW620细胞survivinmRNA的表达
对照组和survivin-asODN处理组均在191bp处出现特异性的survivin条带。空白对照组的SW620细胞survivin mRNA为(0.85±0.06),脂质体-survivin-asODN组和PAMAM-survivin-asODN组分别为(0.56±0.10)和(0.36±0.10),两者均显著低于空白对照组(P＜0.001)。PAMAM-survivin-asODN组survivinmRNA显著低于脂质体-survivin-asOD组,差异有统计学意义(P＜0.01)。
六、SW620细胞survivin蛋白的表达
Survivin蛋白在SW620细胞中有高度表达。空白对照组的SW620细胞survivin蛋白表达为(72.78±6.79),脂质体-survivin-asODN组和PAMAM-survivin-asODN组分别为(35.02±6.28)和(25.36±7.15),两者均显著低于空白对照组(P＜0.001)。PAMAM-survivin-asODN组survivin蛋白显著低于脂质体-survivin-asODN组(P＜0.05)。差异有统计学意义(P＜0.05)。
七、Survivin-asOON对SW620细胞凋亡率的影响
流式细胞仪检测各组SW620细胞凋亡率:经脂质体-survivin-asODN转染复合物和PAMAM-survivin-asODN转染复合物处理SW620细胞48h后,与空白对照组相比,survivinASODN组在Gl期前出现明显的亚二倍体凋亡峰,细胞凋亡率与空白对照组比较差异有统计学意义(P＜0.05)。PAMAM-survivin-asODN复合物的作用更强,两者之间差异有统计学意义(P＜0.05)。
八、结直肠癌SW620细胞裸鼠模型建立
全部裸鼠于腹部皮下接种人结直肠癌细胞系SW620单细胞悬液后均存活并有肿瘤形成,接种点无红肿、破溃,成瘤时间为(15.7±1.14)d。
九、Survivin-asODN对结直肠癌SW620细胞裸鼠模型的体内抑瘤效应
治疗开始前,阴性对照组、脂质体-survivin-asODN组和PAMAM-survivin-asODN组之间肿瘤体积比较,差异无统计学意义(F=0.229,P=0.798,P＞0.05)。
治疗结束时,脂质体-survivin-asODN组肿瘤体积低于治疗前(t=7.918,P=0.000,P＜0.001),PAMAM-survivin-asODN组肿瘤体积低于治疗前(t=11.854,P=0.000,P＜0.001),差异显著。两治疗组与阴性对照组三组间肿瘤体积比较,差异有显著性(F=69.674,P=0.000,P＜0.001)。组间两两比较PAMAM组与脂质体组相比存在显著性差异(P＜0.005),证实PAMAM有更好的抑瘤效果。
治疗结束后处死裸鼠,剥离肿瘤测重量,脂质体-survivin-asODN组和PAMAM-survivin-asODN组肿瘤重量较阴性对照组有不同程度的降低,差异具有统计学意义(P＜0.001)。PAMAM-survivin-asODN治疗组肿瘤重量与脂质体-survivin-asODN治疗组相比,差异具有统计学意义(P＜0.05)。抑瘤率测定可见PAMAM-survivin-asODN治疗组抑瘤率明显高于脂质体-survivin-asODN治疗组,差异在统计学上均有显著性意义(t=48.550,P=0.000,P＜0.01)。
十、裸鼠移植瘤细胞survivin蛋白表达
Survivin蛋白在裸鼠移植瘤细胞中有高度表达。阴性对照组与脂质体-survivin-asODN复合物组、PAMAM-survivinaSODN复合物组比较差异有统计学意义(P＜0.05);PAMAM组survivin蛋白低于脂质体组,两者之间差异有统计学意义(P＜0.05)。
十一、裸鼠移植瘤细胞survivin mRNA表达
通过RT-PCR检测裸鼠移植瘤细胞对照组和survivin-asODN处理组均在191bp处出现特异性的survivin条带。空白对照组细胞的survivin mRNA为(0.85±0.06),脂质体-survivin-asODN组和PAMAM-survivin-asODN组分别为(0.56±0.10)和(0.36±0.10),两者均显著低于空白对照组(P＜0.001)。PAMAM-survivin-asODN组survivin mRNA显著低于脂质体-survivin-asOD组,差异有统计学意义(P＜0.01)。
十二、裸鼠移植瘤细胞的电镜观察
透射电镜下见基因治疗组细胞体皱缩,细胞膜微绒毛消失。细胞核膜发生皱缩或消失,有的胞核核发生碎裂,染色质浓缩、边聚,沿核膜下排列成不同形状和大小的块状,或发生断裂,胞浆内可见多个电子密度增强的核碎片,致密的凋亡小体亦较多见,部分细胞出现细胞器溶解等坏死征象。
十三、survivin-asODN转染复合物治疗后裸鼠重要脏器的组织学观察
为了解PAMAM-survivin-asODN和脂质体-survivin-asODN转染复合物对裸鼠有无毒副作用,取治疗组小裸鼠心、肝、脾、肾、肺等重要脏器行常规病理检查未见明显异常。
结论
1.本实验合成了PAMAM-survivin-asODN转染复合物,呈圆形或类圆形纳米颗粒,具有粒径小,zeta电位高的特点;
2.PAMAM-survivin-asODN转染复合物与脂质体-survivin-asODN的基因包封率、载药率无差异,但对所载基因有缓慢释放作用;
3.PAMAM-survivin-asODN转染复合物中DNA电荷被完全中和,与PAMAM形成稳定复合物。PAMAM对survivin-asODN具有保护作用,可抵御核酸酶的降解作用;
4.PAMAM可将survivin-asODN高效转染入SW620细胞内,其转染效率高于脂质体;
5.Survivin在SW620细胞中有高度表达,PAMAM介导转染针对survivin mRNA的反义寡核苷酸可在mRNA及蛋白两个水平上有效地降低SW620细胞内survivin的表达;
6.经PAMAM-survivin-asODN与脂质体-survivin-asODN转染复合物处理的SW620细胞的增殖均受到抑制,survivin-asODN的作用机制为诱导靶细胞的凋亡:
7.Survivin-asODN对结直肠癌裸鼠移植瘤具有明显的抑制作用,PAMAM-survivin-asODN转染复合物较脂质体-survivin-asODN转染复合物具有更强的抗肿瘤作用;
8.Survivin在裸鼠移植瘤细胞中有高度表达,PAMAM介导转染针对survivinmRNA的反义寡核苷酸可在mRNA及蛋白两个水平上有效地降低裸鼠移植瘤细胞内survivin的表达;
9.survivin-asODN对裸鼠移植瘤细胞的作用机制为诱导靶细胞的凋亡;
10.PAMAM介导survivin-asODN治疗后,荷瘤裸鼠心、肺、肝、肾、小肠等器官无明显组织学变化,提示该系统对荷瘤裸鼠无系统毒副作用。
Background
Colorectal cancer is one of the major diseases that threatenes the health of human,with the incidence increasing in China.The conventional cancer therapies are accompanied by their intrinsic limitations,therefore newly treatment methods are urgently needed.With the development of molecular technology and deep recognition of colorectal cancer,gene therapy represents a novel treatment model in cancer therapy.
More and more data showed that the abnormality of cell apoptosis was very important in cancer development as well as malignant cell proliferation and differentiation hindering.In general condition,apoptosis can destroy DNA-damaged cells and cycle-disordered cells.The proliferation rate of tumor cells was not higher than that of normal cells and the accumulation of tumor cells result from cells escaping apoptosis.The disorder of regulation in apoptosis may well prolong the survival of cells aberrantly,being subject to accumulation of mutation which leads to transformation,and thereby involves in carcinogenesis.So,tumor is not only due to the disorder of cell proliferation and differentiation but also owing to the abnormality of cell apoptosis.
The inducing apoptosis is a new therapy method for tumor and transferring apoptosis-activating gene or apoptosis-inactivating suppressor gene into tumor cells has become one of most intriguing therapeutic strategies.Survivin is the most effective inhibitor factor.
Survivin is a newly discovered member of inhibitor of apoptosis proteins family and play an important role in the regulation of apoptosis.It expresses in human embryonic tissue and takes part in the histological differentiation,organ formation, but almost not in normal differentiated human tissues.Meanwhile,survivin low-expresses in non-proliferation vascular endothelial cells and over-expresses in neogenetic vascular endothelial cells.Therefore,survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.
Antisense oligonucleotides(ASODN) can adjust the message transfer from gene to protein and inhibit the protein expression through specifically combination with target mRNA.ASODN adjust the genetic information transfer on the upper stream, it has good function with lower dosage.ASODN can specifically block the single gene expression without the side effect on normal gene.Survivin only expresses in human embryonic tissue and tumor tissue,it makes the anti-survivin gene therapy a specific and safe therapy.
The important aim of gene therapy is the continually and steadily expression of obtained genetic material in transferred cell.ASODN is a kind of hydrophilic anion ionic polymer,it has poor permeability to biomembrane and be degraded easily by the mucleicacidase,it can't be absorbed and accumulated in the target cell.All this reason restrict the extensive application of ASODN and need a effective carrier.
Gene transfect efficiency is high for traditional virus vector,but the virus posed immunogenic and cytotoxic.The transfection efficiency is low for non-virus vector. Nanometer particle is a kind of natural or synthetic high polymer particle,their diameter are the same with virus among 10-100nm,smaller than normal cell.They can finish the gene therapy through carrying DNA,RNA and oligonucleotide and absorbed by target cell.
Polyamidoamine dendrimer(PAMAM) is a kind of new man-made namometer material.Oligonucleotide/PAMAM Complex will form by electrostatic interaction between surface charged positive electricity of the amidocyanogen group and the DNA main chain charged negative electricity of the phosphoric group,and then the complex can enter the cell by phagocytosis with the help of interaction between cation of the complex surface and the negative electricity of the glycoprotein, phospholipids on the cell surface.The absorb of complex can be promoted and the persistence can be delayed by PAMAM.
In order to elevate the survivin ASOND transfer and strengthen the gene therapy effect,we use PAMAM as a surviving ASODN nanometer carrier,detect its feature and gene transfer rate and release characteristic,observe the killer function of survivin-asODN to colorectal cancer cell SW620 in vitro and vivo.
Objectives
The feasibility of transfecting survivin-asODN mediated by PAMAM and the effects on the expression of survivin and apoptosis to colorectal cancer cells SW620 were investigated.Further,tumors derived from SW620 cells were established in nude mice and the anti-tumor effect of survivin-asODN in vivo was studied.
Methods
1.The preparation of PAMAM antisense gene complex and the investigation of its feature.
PAMAM antisense gene complex was prepared by combining 300μg/L survivin-asODN and 4.06μg/L PAMAM.Similarly,cationic liposome antisense gene complex was prepared as control.The shape of the complex were observed by transmission electron microscope,the size was determined by laser particle size analyzer and the zeta potential was measured by zeta potential analyzer.The encapsulating efficiency,DNA loading level and releasing progress in vitro were determined by ultraviolet spectrophotometer in centrifuge method.
2.The investigation of expression of survivin and apoptosis through transfecting survivin-asODN mediated by PAMAM to colorectal cancer cells SW620 in vitro.
Both of the above gene transfect complexes were used to transfect colorectal cancer cells and transfect efficiencies of survivin-asODN in SW620 cells were measured.Survivin protein expression was checked by Western blot.For both group cells,the apoptosis was assessed by flow cytometry.
3.The investigation of the anti-tumor effect of survivin-asODN mediated by PAMAM to tumors derived from SW620 cells in nude mice.
To establish SW620 carcinoma transplanted subcutaneously in nude mice,4-to 6-week-old female Balb/c nude mice were used as hosts xenografts,SW620 cells were injected subcutaneously into the abdominal region.All tumors were 0.5cm mean diameter and mice were grouped randomly according to treatment regimen.
The tumor and around tissue were injected by Lipolifectmain~(TM2000) and PAMAM (group A:negative control),lipidosome--survivin-asODN(group B) and PAMAM-survivin-asODN(group C).The tumor diameter was measured before and after treatment.The weight of tumors was studied and the growth inhibition rate was calculated after treatment.The change of tunour cells were observed by Transmission Electron Microscope(TEM),the expression of surviving gene in tissue and pathological change in heart,liver,spleen,lung and kidney were observed at the same time.
Results
DNA releasing lasted 14 days for PAMAM,but only 5 days for liposome. Transfect efficiency of PAMAM-survivin-asODN on colorectal cancer cells was higher compared with that of liposome-survivin-asODN(P＜0.05).After the colorectal cancer cells transfected,survivin protein expression was lower(P＜0.05),and apoptosis rate was higher(P＜0.05) compared with control.
1.Observation of the feature and diameter of transfected complex and measurement of zeta potential of transgected complex.
The complex of liposome-survivin-asODN and PAMAM-survivin-asODN were round or round like particle under the TEM,the diameter of PAMAM-survivin-asODN complex was smaller(t=7.874,P=0.000,P＜0.001) compared with that of liposome-survivin-asODN complex,but zeta potential was higher(t=4.158,P=0.002,P＜0.01).
2.Measurement of nuclear acid encapsulating efficiency,loading level and gene releasing in vitro of transfected complex,
There were no significant differences between the two groups in terms of DNA loading level(t=0.178,P=0.863,P＞0.05) and encapsulating efficiency(t=0.582, P=0.573,P＞0.05).There are blasting releasing of gene in the complex of liposome -survivin-asODN in 24～48 hours and decreased quickly.The release of DNA can not be measured in 6 hours.There are blasting releasing of gene in the complex of PAMAM-survivin-asODN in 24～48 hours,and the releasing decrease slowly and last for more than 14 days.
3.Cell transfection and transfection efficiency
24 hours after transfection,green fluorescence could be seen under fluorescence microscope for both groups of cells.48 hours later,the highest transfection efficiency was reached.The fluorescence intensity(46.46±9.84) from liposome group was lower than that from PAMAM group(63.90±10.29),and the difference was statistically significant.(t=2.739,P=0.025,P＜0.05)
4.The growth suppression effect of surviving-asODN to SW620 cells.
There are significant difference between the treatment group of survivin-asODN and the bland control group,the suppression effect of survivin-asODN to SW620 cells was significant(P＜0.001).There growth suppression of cells treated by PAMAM-survivin-asODN and liposome-survivin-asODN,but growth suppression of the PAMAM-survivin-asODN complex was more strong(P＜0.05).
5.The expression of survivin mRNA in SW620 cells.
There were specific surviving strap in 191bp of survivin-asODN treated group and blank control group.The survivin mRNA of SW620 was 0.85±0.06(in blank control group),0.56±0.10(liposome-survivin-asODN group) and 0.36±0.10(PAMAM-survivin-asODN).Compared with the blank control group,there were significant difference in both treated group(P＜0.001).The expression of surviving in PAMAM-survivin-asODN treated group was obviously lower than that in liposome-survivin-asODN treated group(P＜0.01).
6.Effect of Survivin-asODN on survivin protein expression of SW620 cells.
Survivin protein expression from SW620 cells of blank control group was (72.78±6.79),while for the liposome group and PAMAM group were(35.02±6.28)and(25.36±7.15) separately,both of them were significantly lower than the blank control group(P＜0.01);Survivin protein from PAMAM group was significantly lower than that of liposome group(P＜0.05).
7.Effect of Survivin-asODN on the apoptosis rate of SW620 cells.
Compared with empty control group,the cell apoptosis rates from both liposome group and PAMAM group were significantly increased(P＜0.05).While the apoptosis rate of PAMAM group was higher than that of liposome,the difference is statistically significant(P＜0.05).
8.The modeling of nude rate mouse with SW620 tumors.
SW620 cells were injected subcutaneously into the abdominal region of nude mouse and formed tumor successfully.There were no redness and swelling in the injected region.The tumor form time was 15.7±1.14 days.
9.The tumor suppression effect of Survivin-asODN to SW620 cells in nude mouse.
There were no significant difference in gross tumor volume and weight before treatment among blank control group,liposome-survivin-asODN treated group and PAMAM-survivin-asODN treated group(F=0.229,P=0.798,P＞0.05).At the end of treatment,the gross tumor volume was decreased obviously in Lipo-survivin-asODN treated group(t=7.918,P=0.000,P＜0.001) and PAMAM-survivin-asODN treated group(t=11.854,P=0.000,P＜0.001).The gross tumor volume in treated group were significantly lower than that in the blank control group(P＜0.001).Compared with the liposome-survivin-asODN group,the gross tumor volume in PAMAM-survivin-asODN group was significantly lower(P＜0.05).
At the end of treatment,the weight of tumor in both of the treated group was significantly decreased compared with the blank control group(P＜0.001).Compared with the liposome-survivin-asODN group,the weight of tumor in PAMAM-survivin-asODN group was significantly lower(P＜0.05).The tumor suppression effect of PAMAM-survivin-asODN was significantly higher than that of liposome-survivin-asODN(t=48.550,P=0.000,P＜0.01).
10.The surviving protein expression in the transplanted tumor in nude mouse.
There expression of surviving protein in the transplanted tumor in nude mouse was high.There were significant difference between the blank control group and treated group(P＜0.05).The expresson of surviving protein was significantly lower in PAMAM group than that in liposome group(P＜0.05)。
11.The expression of survivin mRNA in transplanted tumor of nude mouse.
There were specific surviving strap in 191bp in blank control group and survivin-asODN treated group through the RT-PCR measurement.The survivin mRNA was 0.85±0.06(blank control group),0.56±0.10 (liposome-survivin-asODN group) and 0.36±0.10(PAMAM-survivin-asODN group). Both them in the treated group were significantly lower than that in blank control group(P＜0.001).It was lower in the PAMAM-survivin-asODN treated group compared with liposome-survivin-asOD treated group(P＜0.01).
12.The observation of transplanted tumor cells in nude mouse under electron microscope.
The tumor cells were crimpled and the microvillus were disappeared after the gene therapy under the transmission electron microscope.The membranes of cell nucleus were crimpled and vanished,some of the cell nucleus were broken,the chromatin were condensed or broken.Pyknotic apoptosis bodys were observed in the tumor cells.
13.The observation of main organ in nude mouse after the survivin-asODN complex treatment.
There were no obvious change in the nude mouse heart,lung,liver,spleen,and kidney observed through routine pathological check after the PAMAM-survivin-asODN and liposome-survivin-asODN treatment.
Conclusion
1.PAMAM-survivin-asODN transfect complex was synthesized in this experiments, the complex was round or round like nanometer particle with small diameter and high potential.
2.There were no significant differences between the PAMAM-survivin-asODN transfect complex and the liposome-survivin-asODN transfect complex in terms of DNA loading level and encapsulating efficiency.But the liposome-survivin-asODN transfect complex can release the target gene slowly and last long time.
3.The electric charge in PAMAM-survivin-asODN transfect complex were compounded stable and completely.The PAMAM can protect the survivin-asODN through resisting the degradation effect by nucleicacidase.
4.PAMAM can transfect survivin-asODN to SW620 cells successfully,its transfect efficiency was high than liposome-survivin-asODN.
5.There were high level expression of survivin in SW620 cells,survivin-asODN transfected by PAMAM decreased the surviving expression through mRNA and protein path.
6.The growth of SW620 treated by PAMAM-survivin-asODN and liposome-survivin-asODN were inhibited through inducing apoptosis.
7.Survivin-asODN obviously suppressed the growth of the SW620 tumor in nude mouse.There were stronger effects in PAMAM-survivin-asODN complex than that in liposome-survivin-asODN complex.
8.There were high level expression of survivin in transplanted SW620 cells in nude mouse,survivin-asODN transfected by PAMAM decreased the surviving expression in transplanted SW620 cells in nude mouse through mRNA and protein path.
9.The growth of transplanted SW620 cells in nude mouse treated by survivin-asODN were inhibited through inducing apoptosis.
10.There were no obvious change in the nude mouse heart,lung,liver,spleen,and kidney observed through routine pathological check after the PAMAM-survivin-asODN treatment,which showed that the PAMAM-survivin-asODN treatment system were safe to the nude mouse.
引文
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