【作者】 王文滪；

【导师】 徐勇； 张志宏；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 目的在选定核干因子(NS)作为“靶基因”的基础上,应用RNA干扰技术,通过建立一种能够在体内和体外将干扰质粒有效、靶向导入前列腺癌细胞的非病毒载体系统,并使干扰片断在前列腺癌细胞中特异性表达,沉默特定癌基因,从而为治疗前列腺癌提供实验基础和理论依据。方法通过构建靶向基因转移非病毒载体Tf-PEG-PEI,分别以PEI和Tf-PEG-PEI将pEGFP-C1质粒及构建的以PSMAe/p为驱动序列的EGFP干扰质粒转入体外培养高表达PSMA的人前列腺癌细胞系LNCap中,通过荧光显微镜观察各组荧光表达,并应用实时定量PCR(real time PCR)和western blot检测EGFP基因和蛋白表达变化,并以不表达PSMA的人前列腺癌细胞系PC-3、不表达PSMA的人膀胱癌细胞系T24及人胚肾HEK293进行对照实验,从而证实PSMAe/p驱动干扰片断的细胞特异性和Tf-PEG-PEI高效靶向基因转移的能力。以功能基因shNS更换EGFP干扰质粒pPSMAe/p-shEGFP-poly(A)质粒中的shEGFP,重构针对NS的干扰质粒,利用LNCaP及PC-3进行细胞实验,以Tf-PEG-PEI为载体将质粒转入细胞,检测细胞的形态、生长情况以及NS基因和蛋白的变化,进一步证实PSMAe/p驱动干扰片断的细胞特异性及Tf-PEG-PEI的高效基因转移能力。从而为后续的研究提供必要的体外细胞实验依据和理论基础。结果荧光显微镜测定结果显示：干扰质粒与pEGFP-C1质粒共转染LNCaP细胞后,分别以PEI及Tf-PEG-PEI为载体的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)。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,差异有统计学意义(P<0.01)。干扰质粒与pEGFP-C1质粒共转染PC-3、T24和HEK293细胞后各组荧光表达无统计学差异(P>0.05),EGFP mRNA和蛋白表达水平无差异；以PEI为载体组和转染相同质粒以Tf-PEG-PEI为载体组对比,荧光表达及EGFP mRNA和蛋白表达无统计学差异(P>0.05),但Tf-PEG-PEI为载体组细胞形态较好,存活率较高,转染效率稍高。前列腺癌细胞系LNCaP和PC-3中均表达NS蛋白；在将干扰质粒pPSMAe/p-shNS-poly(A)转染入LNCaP细胞后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。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。结论Tf-PEG-PEI具有高效靶向基因转移的能力,可将体外的基因片段高效转入体外培养细胞中,且经PEG修饰后细胞毒性大大降低,是一个安全、高效、的RNA干扰输送系统。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、T24细胞和HEK293细胞中未能有效驱动shRNA转录；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】 ObjectiveBased on selection of NS as "target gene",use RNA interference techniques, by creating a non-viral vector system to transfer the interfere plasmid effectively and targetly into the prostate cancer cell in vivo and vitro, so interfere sequence can express specitly in the prostate cancer cell, silencing the specific cancer gene, which provided experimental and theoretical basis for the treatment of prostate cancer.MethodsBy constructing targeting gene transfect non-viral vector Tf-PEG-PEI,using PEI and Tf-PEG-PEI to transfer plasmid pEGFP-C1 and recombinant plasmid targeting interference EGFP which are driven and regulated by prostate specific membrane antigen promoter and enhancer(PSMAe/p) into human prostate cancer cell lines LNCaP which highly express PSMA, then the suppression effect of EGFP was assayed by fluorescence microscope.The mRNA and protein levels of EGFP were detected by real time RT-PCR and Western blot in each group cells.Then use cells which does not express PSMA as comparation,including human prostate cancer cell line PC-3,human bladder cancer cell line T24 and human embryo kidney 293 cell(HEK293).So to demonstrate the cellular specificity of shRNA transcription targeting gene driven by PSMAe/p and the high gene transfer ability of Tf-PEG-PEI. Using shRNA sequence targeting NS to replace the shRNA sequence targeting EGFP of recombinant plasmid pPSMAe/p-shEGFP-poly(A) to construct the new recombinant plasmid,then transfect it into human prostate cancer cell lines LNCaP and PC-3 by Tf-PEG-PEI to detect the changes of cell morphology, cell cycle, proliferation ability,and the expression of NS gene and protein. To further demonstrate the cellular specificity of shRNA transcription targeting gene driven by PSMAe/p and the high gene transfer ability of Tf-PEG-PEI. ResultsFluorescence microscopy showed that:after interference plasmid and pEGFP-C1 plasmid were cotransfected into LNCaP cells, the interference group’s expression of fluorescence decreased which use PEI and Tf-PEG-PEI as a carrier respectively, There were significant differences compared with the control group (P<0.01).The expression of EGFP mRNA and protein in interference group compared with the control group were decreased, the difference was statistically significant (P<0.01). After interference plasmid and pEGFP-C1 plasmid were cotransfected into PC-3,T24 and HEK293 cells, there were no significant difference in fluorescence expression between each groups (P>0.05).EGFP mRNA and protein levels were no significantly difference. Compare the groups that transfer the same plasmid using PEI or Tf-PEG-PEI as carrier, there were no significant difference (P> 0.05)on fluorescence, EGFP mRNA expression and EGFP protein expression, but Tf-PEG-PEI group is better in mprphology, survival, slightly higher transfection efficiency. Both prostate cancer cell lines LNCaP and PC-3 express the NS protein. After the interference plasmid pPSMAe/p-shNS-poly (A) was transfected into LNCaP cells, the level of NS gene expression 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 was discounted 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.ConclusionsTf-PEG-PEI has a high capacity of targeted gene transfer ability, it can transfecte vitro gene into cultured cells efficiently, and modified by PEG greatly reduced its toxicity, is a safe, efficient RNA interference delivery systems. PSMAe/p can drive shRNA’expression specialy in LNCaP cells which express PSMA, but can not in the PC-3 cells, T24 cells and HEK293 cells. 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