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目的:采用抗人血管内皮生长因子(vascular endothelial growth factor,VEGF)发卡状核酶基因转染肝癌细胞,观察核酶对VEGF表达及肿瘤生长的影响。方法:采用脂质体介导的方法,将人工合成的抗VEGF发卡状核酶基因转染肝癌细胞SMMC-7721,同时制备空载体和细胞对照,经G418筛选获得阳性克隆,基因组水平鉴定核酶在细胞中的表达,半定量RT-PCR法和免疫组化法观察核酶对SMMC-7721细胞VEGF表达的影响。接种各组细胞于裸鼠皮下,观察瘤体体积大小和质量,免疫组化法观测各组肿瘤微血管变化和VEGF的表达。结果:核酶基因成功导入到肿瘤细胞中,转基因细胞的增殖速率明显下降(P<0.01),转染核酶组细胞VEGF水平明显下降(P<0.01)。移植瘤成瘤率和生长速度减慢(P<0.01),肿瘤组织VEGF表达和血管形成减少(P<0.01)。结论:抗人VEGF发卡状核酶基因在体内、体外均可抑制肝癌VEGF表达,通过减少血管形成抑制肿瘤生长,为进一步开展肝癌血管靶向基因治疗提供实验依据。
Objective: To investigate the effect of ribozyme on VEGF expression and tumor growth by transfecting hepatoma cells with vascular endothelial growth factor (VEGF) hairpin ribozyme gene. Methods: Liposome-mediated method was used to transfect artificial anti-VEGF hairpin ribozyme gene into SMMC-7721 hepatoma cells. Meanwhile, empty vector and cell control were prepared. Positive clones were screened by G418, and ribozyme was identified by genomic level The expression of VEGF in SMMC-7721 cells was detected by semi-quantitative RT-PCR and immunohistochemistry. All the cells were inoculated subcutaneously in nude mice to observe the size and quality of tumor volume. The changes of tumor microvessels and VEGF expression in each group were observed by immunohistochemistry. Results: The ribozyme gene was successfully introduced into tumor cells. The proliferation rate of transgenic cells was significantly decreased (P <0.01). The VEGF level of transfected ribozyme group was significantly decreased (P <0.01). The rate of tumorigenesis and growth of the xenografts decreased (P <0.01), and the expression of VEGF and angiogenesis decreased (P <0.01). Conclusion: The anti-human VEGF ribozyme gene can inhibit the expression of VEGF in hepatocellular carcinoma in vitro and in vivo, and inhibit tumor growth by reducing the formation of blood vessels, providing an experimental basis for further gene therapy targeting hepatocellular carcinoma.