靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响

目的 构建携带促血管生成素2-小干扰RNA（Ang2-siRNA）慢病毒载体,观察其对恶性黑色素瘤细胞中Ang2基因表达的干扰作用.方法 将经XbaⅠ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒（pNL-EGFP）载体与pSilencer 1.0-U6启动子-促血管生成素2-小干扰RNA（pSilencer 1.0-U6-Ang2-siRNA）重组质粒连接,产生加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅰ（pNL-EGFP-U6-Ang2-Ⅰ）、加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅱ（pNL-EGFP-U6-Ang2-Ⅱ）慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒、水疱性口炎病毒G蛋白（pVSVG）包膜质粒和pHelper包装质粒共转染293T细胞,产生pNL-EGFP-U6-Ang2-Ⅰ、pNL-EGFP-U6-Ang2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Ang2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Ang2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.0×103/ml.实时荧光定量RT-PCR结果显示：Ang2-siRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Ang2基因的表达（P＜0.05）.结论 成功构建了Ang2-SiRNA慢病毒载体,体外研究显示Ang2-SiRNA慢病毒载体能抑制恶性黑色素瘤细胞中Ang2 mRNA的表达,为下一步进行裸鼠恶性黑色素瘤移植瘤生长的干预实验奠定基础,为肿瘤的基因治疗提供实验依据.

Construction of Recombinant Lentiviral Vector of siRNA for Ang2 and the Effect of the Expression of Ang2 Gene in Malignant Melanoma Cells

Objective To construct lentivectors carrying Ang2-small interfering RNA （siR- NA）, which was used to inhibit Ang2 expression in malignant melanoma cells. Methods Plasmid pNL-EGFP and recombinant plasmid pSilencer 1. O-U6-Ang2-siRNA were digested with XbaI and ligated into ~ Plasmids a target lentiviral transfer plasmid of pNL-EGFP-U6-Ang2- I or pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- II were pNL-EGFP-U6-Ang2- II constructed successful- ly. Lentiviral vector systems of three plasmids were constituted using pNL-EGFP-U6-Ang2- I （pNL- EGFP-U6-Ang2-II ）, pVSVG and pHelper. The lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- I1 lentivirus. Superuatant was col- lected to determine the titer. Malignant melanoma cells were transduced with both lentiviruses. Real- time RT-PCR was conducted to examine inhibitory efficiency, Results The recombinant lentiviral vectors of Ang2-RNAi were constructed successfully, as confirmed by restriction enzyme digestion and sequencing. Lentiviral titer was up to 8.0 x 103/ml. Real-time RT-PCR demonstrated that the lentiviral vectors of Ang2-RNAi infect malignant melanoma cells and inhibit the expression of Ang2 genes in malignant melanoma cells （ P 〈 0. 05 ） . Conclusion Lentivector carrying Ang2-siRNA was constructed successfully and it was able to inhibit the expression of Ang2 gene in vitro significantly. The study established preliminary research on the inhibition of the growth of malignant melanoma xenografts in nude mice and provides the experimental evidence for tumor gene therapy in future.