【作者】 孙建涛；

【导师】 徐勇； 张志宏；

【作者基本信息】 天津医科大学 ， 外科学， 2008， 博士

【摘要】 第一部分靶向干扰EGFP的shRNA质粒表达载体的构建和鉴定目的采用不同终止信号构建前列腺特异性膜抗原增强子╱启动子（PSMA e/p）调控的靶向EGFP的shRNA真核表达重组质粒pPSMAe/p-shEGFP-poly（A）,pPSMAe/p-shEGFP-minipoly（A）的pPSMAe/p-shEGFP-poly（U）。方法分别使用pSilencer^TM4.1-CMV neo质粒中RNA聚合酶Ⅱ启动子的终止信号poly（A）,minipoly（A）（AAUAAA六聚体）,RNA聚合酶Ⅲ启动子终止信号poly（U）（TTTTTT）为终止信号,采用分子克隆技术将人工合成的含靶向EGFP的shRNA（EGFP-shRNA）及终止信号序列的DNA片段定向克隆入SalⅠ、BamH双酶切的含有PSMA增强子/启动子基因序列的质粒载体pPSMAe/p上。重组质粒经菌液PCR、限制性内切酶酶切和测序进行鉴定。结果通过菌液PCR,质粒酶切和测序鉴定,人工合成的DNA序列成功插入载体pPSMAe/p上,插入目的基因的真核表达载体的酶切图谱和测序结果与预期一致。结论成功构建了采用不同终止信号,以前列腺特异性膜抗原增强子/启动子驱动的靶向干扰EGFP的shRNA真核质粒表达载体pPSMAe/p-shEGFP-poly（A）,pPSMAe/p-shEGFP-minipoly（A）pPSMAe/p-shEGFP-poly（U）。第二部分PSMAe/p驱动shRNA靶向干扰EGFP的细胞特异性研究目的探讨前列腺特异性膜抗原增强子、启动子（PSMAe/p）驱动shRNA转录效果和细胞特异性,比较何种终止序列更有效。方法通过脂质体Lipofectamine^TM2000介导,将各重组干扰质粒与pEGFP-C1质粒共转染高表达PSMA的人前列腺癌细胞系LNCaP、不表达PSMA的人前列腺癌细胞系PC-3、不表达PSMA的人膀胱癌细胞系EJ和人胚肾HEK293细胞,采用荧光显微镜,流式细胞仪,半定量逆转录聚合酶链反应（RT-PCR）和Western blot检测EGFP在各组细胞中的干扰效果。结果荧光显微镜和流式细胞仪测定结果显示:各干扰质粒与pEGFP-C1质粒共转染LNCaP后,各干扰组荧光表达均有不同程度减少,与空载体组相比均有统计学差异（P＜0.01）,pPSMAe/p-shEGFP-poly（A）组减少与pPSMAe/p-shEGFP-minipoly（A）组相比有统计学差异（P＜0.05）,和pPSMAe/p-shEGFP-poly（U）组相比有显著统计学差异（P＜0.01）。各干扰载体与pEGFP-C1质粒共转染PC-3、EJ和HEK293细胞后各组荧光表达无统计学差异（P＞0.05）,EGFP mRNA和蛋白表达水平无明显差异;LNCaP细胞各干扰组中EGFP mRNA和蛋白表达与对照组相比均有不同程度下降,以pPSMAe/p-shEGFP-poly（A）组最为明显。结论PSMAe/p能在高表达PSMA的LNCaP细胞中特异性驱动shRNA转录,而在不表达PSMA的PC-3细胞、EJ细胞和HEK293细胞中未能有效驱动shRNA转录;在三种终止信号中poly（A）信号终止转录最有效。第三部分PSMAe/p驱动shRNA靶向干扰核干因子治疗前列腺癌的研究目的进一步探讨PSMAe/p驱动shRNA靶向干扰NS基因的细胞特异性;通过RNA干扰观察NS基因沉默对前列腺癌细胞和裸鼠移植瘤生长的影响,探讨NS基因与前列腺癌恶性增殖的关系,为前列腺癌的基因治疗探索新的靶基因和靶向策略。方法免疫组化检测人前列腺癌细胞系LNCaP和PC-3中NS表达;合成以poly（A）为终止信号的针对NS基因的shRNA对应模版的DNA序列,定向克隆入SalⅠ、BamHⅠ双酶切的pPSMAe/p-shEGFP-poly（A）的载体中,构建重组质粒pPSMAe/p-shNS-poly（A）。脂质体Lipofectamine^TM2000介导重组质粒转染入人前列腺癌LNCaP和PC-3细胞系中。采用半定量RT-PCR和Western blot技术检测干扰后NS mRNA和蛋白水平表达的变化,应用细胞计数检测细胞增殖情况,应用流式细胞仪检测细胞周期、细胞凋亡变化情况。以LNCaP细胞构建裸鼠前列腺癌移植瘤模型,肿瘤局部多点注射重组干扰质粒,观测治疗过程中肿瘤体积和测量最终重量,免疫组织化学染色法观察各组中NS蛋白表达情况。结果前列腺癌细胞系LNCaP和PC-3中均表达NS蛋白;经过酶切及测序鉴定成功构建重组质粒pPSMAe/p-shNS-poly（A）。在LNCaP细胞中NS基因表达水平下调,细胞增大,胞膜边缘突起增多,更趋向于分化;S期细胞的百分率降低,G1期的百分率升高;细胞的体外增殖速率明显降低,细胞凋亡增多。在PC-3细胞中NS基因表达无明显下调,细胞周期和增殖能力无明显变化。LNCaP细胞移植瘤生长速度、肿瘤体积和最终重量也明显降低。结论PSMAe/p驱动shRNA靶向干扰NS基因具有细胞特异性,使用细胞特异性启动子是实现前列腺癌靶向基因治疗的有效策略;NS基因可能是前列腺癌LNCaP细胞周期中G1/S检查点一个重要的调节因子,在LNCaP细胞恶性增殖中发挥重要作用,NS可能是前列腺癌基因治疗的理想靶基因之一。更多还原

【Abstract】 PartⅠConstruction and verification of shRNA recombinant plasmid targeting interference EGFPObjective Utilizing three different termination signals to construct three eukaryotic expression recombinant plasmids targeting interference EGFP（enhanced green fluorescent protein）which are driven and regulated by prostate specific membrane antigen promoter and enhancer（PSMAe/p）.Methods The three designed interference sequences targeting EGFP using poly（A）which is the termination signal of plasmid pSilencer^TM4.1-CMV neo,minipoly（A）（AAUAAA hexamer）, poly（U）（TTTTTT）as termination signals respectively were cloned into the pPSMAe/p vector,which was ligated after SalⅠand BamHⅠdigestion.Poly（A）and minipoly（A）termination signals belong to RNA polymeraseⅡpromoter,whereas poly（U）belongs to RNA polymeraseⅢpromoter.The recombinant plasmids were verified by PCR of the bacterium liquids,restriction enzyme digestion and gene sequencing.Results The artificial DNA sequences were successfully inserted into the pPSMAe/p carrier respectively.The enzyme digesting spectra and sequencing results of the recombinant plasmids were correct.Conclusion The eukaryotic expression recombinant plasmids utilizing different termination signals targeting EGFP:pPSMAe/p-shEGFP-poly（A）,pPSMAe/p-shEGFP-minipoly（A）,pPSMAe/p-shEGFP-poly（U） were successfully constructed.PartⅡStudy of Cell specificity of shRNA targeting EGFP driven by PSMA enhancer and promoterObjective To study the effects and cell specificity of prostate specific membrane antigen promoter and enhancer（PSMAe/p）in driving short hairpin RNA （shRNA）transcription and explore which terminator signal is more effective. in human prostate cancer cell lines LNCaP and PC-3.The designed shRNA sequence targeting NS with poly（A）termination signal was cloned into the pPSMAe/p -shEGFP-poly（A）vector,which was ligated after Sal I and BamH I digestion. Recombinant plasmid pPSMAe/p-shNS-poly（A）was transfected into human prostate cell lines LNCaP and PC-3 with Lipofectamine^TM2000.The mRNA and protein levels of NS were detected by semi-quantitative RT-PCR and Western blot.The changes of cell morphology,cell cycle,proliferation ability and apoptosis were also studied after down-regulating the NS gene level.LNCaP cells were inoculated into nude mice to establish xenograft tumor model.Xenograft tumors were injected with recombinant plasmid,investigating the volume of tumors during therapy and measuring the final weight of tumors,Immunohistochemistry was used to detect the expression of NS in each group.Results Both LNCaP cells and PC-3 cells express NS protein.We can determine the successful construction of recombinant plasmid pPSMAe/p-shNS-poly（A）by enzyme digestion and sequence analysis.The expression level of NS gene in LNCaP cells was downregulated,cells became larger and showed more pseudopodia,having a tendency to differentiate,the detection of cell cycle showed a decrease of S stage and an increase of G1 stage,cell proliferation ability in vitro and tumorigenesis ability in nude mice were discounted,the tumor volume and final weight were also decreased after knocking down NS gene.The downregulation of NS gene expression level wasn’t conspicuous in PC-3 cells,cell cycle and cell proliferation ability didn’t change obviously.Conclusion The shRNA transcription targeting NS gene driven by PSMAe/p has cellular specificity.Utilizing cell specific promoter is a effective strategy for targeting gene therapy in prostate cancer.NS may act as an important G1/S regulator to regulate the malignant proliferation of LNCaP cells.NS gene may serve as an ideal therapeutic target for prostate cancer.更多还原