STAT3-siRNA慢病毒表达载体的制备及其在SW480细胞中的表达

Preparation and expression of STAT3-specific lentiviral expression vector in colorectal cancer cell SW480

【目的】通过慢病毒感染SW480细胞观察RNAi对细胞内STAT3基因表达的影响。【方法】制备STAT3干扰质粒,与其它3种质粒共同转染至293T细胞中组装为STAT3-siRNA慢病毒表达载体。流式测定病毒滴度后选择最适滴度感染SW480细胞并进行分选。Real-time PCR及Western blotting分析该慢病毒在SW480细胞中对于STAT3基因表达的影响。【结果】测序结果说明制备的慢病毒表达载体符合实验预期。该siSTAT3慢病毒感染SW480细胞后,流式细胞仪检测发现SW480细胞凋亡比率增加,Real-time PCR检测发现慢病毒对于STAT3 mRNA表达量的干扰效率为78.68%,Western blotting检测发现STAT3蛋白表达量下降,与对照组相比差异有统计学意义(P<0.05)。【结论】本研究制备的STAT3-siRNA慢病毒表达载体,能够有效干扰结直肠癌SW480细胞株STAT3表达。

【Objective】To prepare lentivirus expression vector for RNA interference（RNAi） of human STAT3 and observe its effect on the expression of STAT3 in SW480.【Methods】Prepared the interfering plasmid targeted to STAT3, and packaged it with other 3 plasmids into STAT3-siRNA lentivirus expression vector in 293T cells. Selected the optimal virus titer to infect human colorectal cancer cell SW480 and sorted them by the flow cytometry. The expression of STAT3 in SW480 was detected by Real-time PCR and Western blotting.【Result】Result of sequencing proved that the lentivirus expression vector obtained was coincided with the experiment expectancy. The apoptosis ratio of SW480 cells was increased after it had been infected by siSTAT3 lentivirus. The expression of STAT3 mRNA and protein was significantly decreased as compared with GFP group（P 0.05）, especially the expression of STAT3 mRNA was reduced by 78.68% contrasted with negative control group.【Conclusion】This study completed the package of lentivirus vector encoding STAT3-siRNA, which can interfere the expression of STAT3 effectively in human colorectal cancer cell SW480.