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目的:构建TGFBR2基因的RNA干扰慢病毒载体,为研究该基因在结肠癌细胞中的作用奠定基础。方法:合成TGFBR2基因特异性RNAi靶序列,与pcDNATM6.2-GW/EmGFP-miR载体连接,构建干扰载体并鉴定。与TGFBR2质粒共转染HEK293细胞,qRT-PCR和Western blot方法筛选最佳干扰序列,感染SW480细胞。10ng/ml TGF-β1和20ng/ml TNF-a共作用TGFBR2干扰后的SW480细胞和未干扰组细胞72h后,通过细胞侵袭试验计算细胞侵袭数目。结果:成功构建TGFBR2的RNA干扰载体,第4组序列效果最佳,感染SW480后,TGFBR2基因的mRNA表达抑制率为78.45%。TNF-a和TGF-β1处理干扰前后的SW480细胞,通过细胞侵袭试验显示各组细胞迁移的数目:干扰组为89.3±7.9,未干扰组为15.1±8.2,干扰组细胞明显多于对照组(P<0.01)。结论:成功构建人TGFBR2基因特异性的慢病毒干扰载体,并建立稳定的细胞株,为预防和干预结肠癌的早期转移奠定基础。初步提示在TGF-β1和TNF-a因子存在的炎症微环境中,TGFBR2基因突变的结肠癌细胞更具有侵袭性。
Objective: To construct RNA interference lentiviral vector of TGFBR2 gene and lay the foundation for the study of its function in colon cancer cells. Methods: TGFBR2 gene specific RNAi target sequence was synthesized and ligated with pcDNATM6.2-GW / EmGFP-miR vector to construct the interference vector and identified. HEK293 cells were co-transfected with TGFBR2 plasmid, and the optimal interference sequences were screened by qRT-PCR and Western blot to infect SW480 cells. After co-administration of 10ng / ml TGF-β1 and 20ng / ml TNF-α for 24 hours, the invasiveness of SW480 cells and non-interfering cells were evaluated by cell invasion assay. Results: The RNA interference vector of TGFBR2 was constructed successfully, and the sequence of the fourth group was the best. After transfected with SW480, the mRNA expression of TGFBR2 was inhibited by 78.45%. The number of cell migration in each group was 89.3 ± 7.9 in the interference group, 15.1 ± 8.2 in the non-interference group, and more in the interference group than in the control group P <0.01). CONCLUSION: The human TGFBR2 gene-specific lentiviral vector was successfully constructed and a stable cell line was established, which laid the foundation for the prevention and intervention of early metastasis of colon cancer. Preliminary indications Colon cancer cells with TGFBR2 mutations are more aggressive in the inflammatory microenvironment in which TGF-β1 and TNF-α exist.