靶向DNA聚合酶的人工microRNA用于肝癌基因治疗的研究

发布时间：2018-05-29 05:36

本文选题：靶向 + 聚合　；参考：《浙江省医学科学院》2015年硕士论文

【摘要】：目的：肝细胞肝癌(hepatocelluar carcinoma, HCC)是致死率很高的恶性肿瘤之一,目前的治疗手段效果有限。新兴的基因治疗给肿瘤患者带来了新的希望,可以通过组织特异性启动子调控导入细胞的治疗性基因靶向表达。然而,肝癌基因治疗的靶点多集中于肝癌发生发展过程中的相关基因,只能对相应类型的肿瘤起效。在本研究中,我们选择DNA复制环节这一所有细胞增殖的关键步骤作为治疗靶点,来抑制肿瘤细胞生长。方法：本课题构建了包含HCC特异性甲胎蛋白(a-fetoprotein, AFP)增强子的重组AFP启动子调控串联连接的分别靶向DNA聚合酶α,δ,ε的人工microRNA和重组活化Caspase3基因的重组腺病毒Ad/AFP-Casp-AFP-amiR,同时构建仅有重组AFP启动子的重组腺病毒Ad/AFP作为空白病毒对照。结果：体外实验：重组腺病毒以MOI 50感染AFP强阳性的HCC细胞HepG2、AFP弱阳性的HCC细胞Hep3B和AFP阴性的正常肝细胞HL7702, Ad/AFP-Casp-AFP-amiR能够抑制HepG2和Hep3B DNA聚合酶α,δ,ε的表达,阻滞细胞周期在G1期；诱发HepG2和Hep3B细胞凋亡、抑制细胞增殖。Ad/AFP-Casp-AFP-amiR对肝癌细胞的抑制效应与AFP的表达水平正相关。Ad/AFP-Casp-AFP-amiR对正常肝细胞HL7702没有作用。体内实验：建立裸鼠Hep3B移植瘤模型,瘤内注射Ad/AFP-Casp-AFP-amiR,未能观察到明显的抑瘤效应,但能够诱发细胞凋亡。结论：Ad/AFP-Casp-AFP-amiR有望成为新的肝癌基因治疗药物,通过AFP启动子特异性抑制AFP阳性的肝癌细胞DNA聚合酶α,δ,￡表达,阻遏肝癌细胞生长,诱发肝癌细胞凋亡。这一方法基于抑制细胞增殖的关键步骤DNA复制,不局限于某一种基因异常表达的HCC,为肝癌的基因治疗提供了新的思路。
[Abstract]:Objective: hepatocellular carcinoma (HCC) is one of the malignant tumors with high mortality. The new gene therapy has brought new hope to tumor patients, which can regulate the target expression of therapeutic gene by tissue specific promoter. However, the target of gene therapy for liver cancer is mainly related to the occurrence and development of liver cancer, which can only work on the corresponding types of tumor. In this study, we selected DNA replication, a key step of cell proliferation, as a therapeutic target to inhibit the growth of tumor cells. Methods: in this study, the recombinant AFP promoter containing HCC specific alpha-fetoprotein (AFP) enhancer was constructed to regulate the tandem linkage of DNA polymerase 伪, 未, 蔚, and the recombinant adenovirus Ad-AFP-Casp-AFP-amiR was constructed, and the recombinant adenovirus Ad-AFP-Casp-AFP-amiR was constructed respectively, which was targeted at DNA polymerase 伪, 未, 蔚, and the recombinant adenovirus Ad-AFP-Casp-AFP-amiR, respectively. The recombinant adenovirus Ad/AFP with only recombinant AFP promoter served as a blank virus control. Results: in vitro, recombinant adenovirus infected AFP strongly positive HCC cell HepG2Afefefefefefefefefefefefefefefefefefefefefefe-positive HCC cells with MOI 50 could inhibit the expression of HepG2 and Hep3B DNA polymerase 伪, 未, 蔚 and arrest the cell cycle in G1 phase, and Ad/AFP-Casp-AFP-amiR could inhibit the expression of HepG2 and Hep3B DNA polymerase 伪, 未, 蔚 in normal hepatocytes. The apoptosis of HepG2 and Hep3B cells was induced, and the inhibitory effect of Ad-r-AFP-Casp-AFP-amiR on hepatoma cells was positively correlated with the expression level of AFP. Adr-AFP-Casp-AFP-amiR had no effect on HL7702 of normal hepatocytes. In vivo experiment: Hep3B transplanted tumor model was established in nude mice. Ad-AFP-Casp-AFP-amiR was injected intratumoral into the tumor. No obvious tumor inhibition effect was observed, but apoptosis was induced by the injection of Ad-AFP-Casp-AFP-amiR. Conclusion the AFP promoter can inhibit the expression of DNA polymerase 伪, 未 in AFP positive hepatoma cells, inhibit the growth of HCC cells and induce apoptosis of HCC cells. This method is based on the key step of inhibiting cell proliferation, and is not limited to the abnormal expression of DNA, which provides a new idea for gene therapy of hepatocellular carcinoma.
【学位授予单位】：浙江省医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7;R450

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