【作者】 刘惠宁；

【导师】 林秋华；

【作者基本信息】 中南大学 ， 妇产科学， 2008， 博士

【摘要】 第一章高低侵袭力绒毛膜癌细胞株差异表达基因的筛选和鉴定目的采用基因芯片技术比较高、低侵袭力绒毛膜癌细胞株JEG-3和JAR的基因表达谱,以筛选出与绒毛膜癌侵袭转移相关的基因,为阐明绒毛膜癌侵袭转移的分子机制提供理论依据。方法（1）应用MTT和Matrigel体外侵袭试验检测不同人绒毛膜癌细胞系JEG-3与JAR之间增殖能力和侵袭能力的差异;（2）提取绒毛膜癌细胞株JEG-3和JAR的总RNA,反转录制备杂交探针,运用基因芯片进行杂交,杂交信号用Agilent Scanner扫描,用Imagene软件和Genespring软件分析和处理数据;（3）应用RT-PCR技术检测小窝蛋白-1（CAV-1）和血管内皮细胞生长因子B（VEGF-B）在JEG-3和JAR绒毛膜癌细胞株中的表达情况,进而验证基因芯片结果的可信度。结果（1）JEG-3和JAR细胞之间增殖能力的差异无显著性（P＞0.05）,但JEG-3细胞的侵袭能力显著高于JAR细胞（P＜0.05）;（2）对绒毛膜癌JEG-3和JAR细胞株的基因表达谱分析,发现两者表达差异显著的基因有742个,其中在JEG-3细胞中321个基因表达上调,422个基因表达下调,上调基因中与细胞侵袭转移能力有关的基因有51个,其中差异最显著的前五位分别是纤维连接蛋白（FN）、基质金属蛋白酶-2（MMP-2）、尿激酶型纤溶酶原激活因子（uPA）、小窝蛋白-1（CAV-1）和血管内皮细胞生长因子B（VEGF-B）;（3）CAV-1和VEGF-B mRNA在JEG-3细胞中的表达水平显著高于在JAR细胞中的表达水平,结果与基因芯片结果一致。结论（1）多个侵袭转移相关基因的表达在绒毛膜癌JEG-3和JAR细胞间存在着差异;（2）基因芯片技术能有效地分析各细胞系的基因表达谱、为研究绒毛膜癌转移机制提供新的途径。第二章靶向Caveolin-1 shRNA载体的构建和沉默效应观察目的设计合成针对Caveolin-1基因的shRNA质粒载体,转染人绒毛膜癌高侵袭力细胞JEG-3,应用RNA干扰技术抑制JEG-3细胞中Caveolin-1基因,建立可用于研究Caveolin-1基因功能的JEG-3细胞模型。研究Caveolin-1 shRNA转染对人绒毛膜癌高侵袭力细胞株JEG-3中Caveolin-1表达及其对JEG-3细胞在体内、外浸润和转移能力的影响。旨在进一步明确Caveolin-1在人绒毛膜癌进展过程中的作用及机制,探讨将Caveolin-1作为靶分子,运用shRNA技术对人绒毛膜癌进行基因治疗的可行性。方法（1）构建靶向抑制Caveolin-1基因表达的短发夹双链RNA（shRNA）真核表达载体pshRNA-Caveolin-1;（2）采用脂质体转染法将载体导入人绒毛膜癌高侵袭力细胞JEG-3中,利用G-418对转染细胞JEG-3进行稳定表达克隆筛选,获得稳定持续表达shRNA的JEG-3细胞;（3）用RT-PCR和Western Blot方法分别从mRNA和蛋白水平观察RNA干扰对Caveolin-1表达的抑制效应;（4）应用Matrigel体外侵袭试验检测Caveolin-1 shRNA对绒毛膜癌细胞的体外侵袭能力的影响;（5）建立裸鼠皮下异种移植模型,观察Caveolin-1 shRNA对绒毛膜癌细胞的体内侵袭、转移能力的影响。结果（1）成功构建靶向抑制Caveolin-1基因的shRNA载体,并经测序证实;（2）建立可用于研究Caveolin-1基因功能的JEG-3细胞模型;（3）发现转染pshRNA-Caveolin-1的JEG-3细胞中Caveolin-1 mRNA及蛋白质的表达比空白组和阴性对照组JEG-3细胞中Caveolin-1 mRNA及蛋白质的表达均明显下降,差异具有显著性（P＜0.05）;（4）通过Matrigel体外侵袭试验证明抑制Caveolin-1基因在JEG-3细胞中的表达可降低JEG-3细胞的体外侵袭能力（P＜0.05）;（5）在对裸鼠模型研究中发现,抑制Caveolin-1基因在JEG-3细胞中的表达可使裸鼠皮下肿瘤的大小降低（P＜0.05）,肿瘤细胞肺转移的发生率降低（P＜0.05）。结论（1）细胞内表达shRNA可长期敲低靶基因的表达,可建立较理想的基因功能研究细胞模型。（2）Caveolin-1基因与人绒毛膜癌侵袭、转移相关,可能是一种新的肿瘤治疗靶分子。（3）RNA干扰有望为人绒毛膜癌基因治疗提供新策略。第三章靶向VEGF-B shRNA载体的构建和沉默效应观察目的:研究VEGF-B shRNA转染对人绒毛膜癌高侵袭力细胞株JEG-3中VEGF-B表达及JEG-3细胞体内外侵袭、转移、促进血管生成能力的影响。旨在进一步明确VEGF-B在人绒毛膜癌进展过程中所起的作用及机制,探讨将VEGF-B作为靶分子,运用shRNA技术对人绒毛膜癌基因治疗的可行性。方法:（1）构建靶向抑制VEGF-B基因表达的短发夹双链RNA（shRNA）真核表达载体pshRNA-VEGF-B;（2）采用脂质体转染法将载体导入人绒毛膜癌高侵袭力细胞JEG-3中,利用G-418对转染细胞JEG-3进行稳定表达克隆筛选,获得稳定持续表达shRNA的JEG-3细胞;（3）用RT-PCR和Western Blot方法分别从mRNA和蛋白水平观察RNA干扰对VEGF-B表达的抑制效应;（4）应用Matrigel体外侵袭试验检测VEGF-B shRNA对绒毛膜癌细胞的体外侵袭能力的影响;（5）建立裸鼠皮下异种移植模型,观察VEGF-B shRNA对JEG-3细胞侵袭转移能力的影响;（6）采用免疫组织化学技术,检测移植瘤组织中VEGF-B的表达情况以及CD₃₄标记的MVD表达情况。结果:（1）成功构建靶向抑制VEGF-B基因的shRNA载体,并经测序证实;（2）建立可用于研究VEGF-B基因功能的JEG-3细胞模型;（3）发现转染pshRNA-VEGF-B的JEG-3细胞中VEGF-B mRNA及蛋白质的表达比空白组和阴性对照组JEG-3细胞中VEGF-B mRNA及蛋白质的表达均明显下降,差异具有显著性（P＜0.05）;（4）通过Matrigel体外侵袭试验证明抑制VEGF-B基因在JEG-3细胞中的表达可降低JEG-3细胞的体外侵袭能力（P＜0.05）;（5）在对裸鼠模型研究中发现,抑制VEGF-B基因在JEG-3细胞中的表达可使裸鼠皮下肿瘤的大小降低（P＜0.05）,肿瘤细胞肺转移的发生率亦有降低（P＜0.05）;（6）VEGF-B在空白组和阴性对照组移植瘤中的表达较转染组高（P＜0.05）,MVD在空白组和阴性对照组移植瘤中高表达,在转染组移植瘤中表达降低（P＜0.05）;（7）相关性分析显示VEGF-B蛋白表达水平与绒毛膜癌血管生成能力之间成正相关。结论:（1）细胞内表达shRNA可长期敲低靶基因的表达,可建立较理想的基因功能研究细胞模型。（2）VEGF-B在人绒毛膜癌中的表达与MVD呈正相关,提示微血管的形成与VEGF-B的调控有关,MVD增高是人绒毛膜癌发生侵袭和远处转移的基础。（3）由shRNA介导的RNAi技术可能是一种有效的肿瘤基因治疗新方法。更多还原

【Abstract】 PartⅠIdentification of diferentially expressed genes in human choriocarcinoma cell lines with different metastasis potentialObjective In order to obtain choriocarcinoma metastasis-associated genes,the transcriptional profiles of human choriocarcinoma cell lines JEG-3(highly metastatic) and JAR(lowly metastatic) were compared using cDNA microarrayMethods(1) The different invasion and proliferation ability between JEG-3 and JAR cell lines was proved by Matrigel invasion assay and MTT assay in vitro;(2) Total RNA of human choriocarcinoma JEG-3 cells and JAR cells was extracted, purified to mRNA and then reversely transcripted to cDNA probe respectively.The cDNA probe of JEG-3 was labelled with Cy3 and the cDNA probe of JAR was labelled with Cy5.The two samples were hybridized with the cDNA microarray.The hybridization signals were scanned by Agilent Scanner and obtained data were analyzed using Imagene software and Genespring software.(3) In order to confirm the quality of gene chip,Reverse transcriptase polymerase chain reaction(RT-PCR) was carried out to determine CAV-1 and VEGFB gene expression.Results(1) We proved that the invasion ability of JEG-3 cell line was stronger than that of JAR cell line(P＜0.05),but the difference of proliferation ability between JAR and JEG-3 is not obvious(P＞0.05);(2) 743 differential expression genes were found between JEG-3 and JAR cell lines by analyzing gene expression profile.There were 321 upregulated genes and 422 downregulated genes in JEG-3 cell line.In upregulated genes,there are 51 genes were correlated with the cell metastasis ability, and FN、MMP-2、uPA、CAV-1 and VEGF-B are the first five genes;(3) We found that the CAV-1 and VEGFB gene in JEG-3 were significantly higher than those in JAR cell lines(P＜0.05),the results of RT-PCR verified the gene chip further.Conclusions(1) There were many differently expressed metastatic genes between JEG-3 and JAR cell lines;(2) The technology of cDNA microarray could analyze effectively gene expression profile of different cell lines,and supply a new approach to study the mechanism of choriocarcinoma metastasis PartⅡConstruction of Caveolin-1 shRNA expressing vector and observation of its gene silencing effectObjective Caveolin-1 gene specific shRNA expressing plasmid was constructed and then transfected into JEG-3 cells so cell model was established to study VEGF-B gene function.Investigate the effects of shRNA transfection on Caveolin-1 expression and the potential of invasion and metastasis in human choriocarcinoma cell lines JEG-3(highly metastatic) in vivo and vitro.To further identify the role and mechanism of Caveolin-1 in the progression of human choriocarcinoma and investigate the feasibility of using gene therapy of shRNA technology to treat human choriocarcinoma with Caveolin-1 as the target molecule.Methods(1) Caveolin-1 gene specific shRNA expressing plasmid was constructed;(2) Caveolin-1 gene specific shRNA expressing plasmid is transfected into JEG-3 cells by lipofectamineTM 2000,non-transfected cells and non-specific shRNA were taken as controls;(3)Inhibitory effect of VEGF-B shRNA was demonstrated by RT-PCR and Western blot;(4) The effect of Caveolin-1 shRNA on invasion in JEG-3 cells in vitro was detected by Matrigel invasion assay;(5) The effects of Caveolin-1 shRNA on potential of invasion and metastasis in JEG-3 cells in vivo were observed using nude mice subcutaneous xenotransplantation model.Results(1) Caveolin-1 gene specific shRNA expressing plasmid was constructed;(2) Caveolin-1 gene specific shRNA expressing plasmid is transfected into JEG-3 cells by lipofectamineTM 2000.Non-transfected cells and non-specific shRNA were taken as controls;(3)Inhibitory effect of Caveolin-1 shRNA was demonstrated by RT-PCR and Western blot,and there was a statistically significant difference comparing with the controls(P＜0.05);(4) Caveolin-1 shRNA transfection significantly downregulated invasion level of JEG-3 cells in vitro(P＜0.05);(5) Caveolin-1 shRNA transfection decreased the size of subcutaneous tumor(P＜0.05),and the pulmonary metastasis incidence is decreased also(P＜0.05).Conclusions(1) Specific shRNA can mediate long-term and stable silencing effects on VEGF-B gene.(2) Caveolin-1 is correlated with cell proliferation,invasion and metastasis of human choriocarcinoma,and it might be a new tumor therapy target molecule.(3) RNAi is expected to supply new strategy for human choriocarcinoma.PartⅢConstruction of VEGF-B shRNA expressing vector and observation of its gene silencing effectObjective To investigate the effects of shRNA transfection on VEGF-B expression and the potential of invasion and metastasis and angiogenesis in human choriocarcinoma cell lines JEG-3(highly metastatic) in vivo.To further identify the role and mechanism of VEGF-B in the progression of human chorioearcinoma and investigate the feasibility of using gene therapy of shRNA technology to treat human choriocarcinoma with VEGF-B as the target molecule.Methods(1)VEGF-B gene specific shRNA expressing plasmid was constructed;(2)VEGF-B gene specific shRNA expressing plasmid is transfected into JEG-3 cells by lipofectamineTM 2000.Non-transfected cells and non-specific shRNA were taken as eontrols;(3)Inhibitory effect of VEGF-B shRNA was demonstrated by RT-PCR and Western blot;(4) The effect of VEGF-B shRNA on invasion in JEG-3 cells in vitro was detected by Matrigel invasion assay;(5)The effects of VEGF-B shRNA on potential of invasion and metastasis in JEG-3 cells in vivo were observed using nude mice subcutaneous xenotransplantation model;(6) Immunohistochemical technology was performed to detect the VEGF-B and MVD(marked with CD34) in transplanted tumor.Results(1)VEGF-B shRNA expressing plasmids were successfully constructed;(2)JEG-3 cell model was established to study VEGF-B gene function;(3)VEGF-B specific shRNA effectively inhibited the expression of VEGF-B at the mRNA and protein levels,and there was a statistically significant difference comparing with the controls(P＜0.05);(4) VEGF-B shRNA transfection significantly downregulated invasion level of JEG-3 cells in vitro(P＜0.05);(5) VEGF-B shRNA transfection decreased the size of subcutaneous tumor(P＜0.05),and the pulmonary metastasis incidence is decreased also(P＜0.05);(6)Strong VEGF-B expression was found in the control transplanted tumor,whereas weak expression in the transfected transplanted tumor,there was statistical difference between the control and the transfected(P＜0.05).High MVD expression was found in the control,whereas low expression in transfected,there was statistical difference between the groups(P＜0.05).There was positive relationship between VEGF-B and MVD expression;(7) The data suggested that there were significantly positive correlations between VEGF-B expression and the angiogenesis of choriocarcinoma.Conclusions(1) Specific shRNA can mediate long-term and stable silencing effects on VEGF-B gene.(2) There is positive relationship between VEGF-B and MVD in human choriocarcinoma indicate the formation of microvessel is regulated by VEGF-B.Increasing MVD is the basis for invasion and metastases in human choriocarcinoma.(3) RNAi technology mediated by shRNA might be an effective tumor gene therapy method.更多还原