论文部分内容阅读
目的 利用RNA干扰(RNAinterference, RNAi)技术, 以突变p53基因为靶基因,构建干扰载体,并观察载体对肿瘤细胞生长周期的影响,为下一步探索肿瘤基因治疗的新途径奠定基础。方法 设计具有小发夹结构的两条DNA序列,经退火成为互补双链,再克隆至干扰载体中,转化TOP10菌株,提取质粒行酶切鉴定,稳定转染PLC/PRF/5细胞,Westernblot检测p53蛋白,MTT法观察细胞生长曲线,流式细胞仪分析细胞周期变化。结果 成功构建针对突变p53的特异性RNA干扰载体,此干扰载体转染PL/PRF/5细胞后,p53蛋白表达水平降低,肿瘤细胞生长速度减慢,G1期细胞分布明显增多。结论 成功构建针对突变p53的RNA干扰载体,此干扰载体可以有效降低p53蛋白表达水平,导致突变p53基因沉默,进而延缓PLC/PRF/5细胞生长速度,阻滞肿瘤细胞停滞于G1期。
OBJECTIVE: To construct interference plasmids by targeting p53 gene by RNA interference (RNAi) technique and to observe the influence of vector on the growth cycle of tumor cells, so as to lay the foundation for further exploring new ways of gene therapy of tumor. Methods Two DNA sequences with small hairpin structure were designed and annealed into complementary double-stranded DNA. The double-stranded DNA was annealed and cloned into the interference vector. The TOP10 strain was transformed into E.coli. The plasmid was digested with restriction endonuclease and stably transfected into PLC / PRF / 5 cells. p53 protein, MTT assay cell growth curve, cell cycle analysis by flow cytometry. Results The specific RNAi vector targeting mutant p53 was successfully constructed. The expression of p53 protein decreased after transfected with PL / PRF / 5 cells, the growth of tumor cells slowed down and the distribution of cells in G1 phase increased significantly. Conclusion The RNA interference vector targeting mutant p53 was successfully constructed. The interference vector could effectively reduce the expression of p53 protein, lead to the silencing of mutant p53 gene, delay the growth of PLC / PRF / 5 cells and arrest the arrest of G1 phase in tumor cells.