SPAG6基因RNAi慢病毒载体的构建及其对SKM-1细胞凋亡的影响  

Construction of lentiviral vector for SPAG6 RNA interference and its influence on apoptosis of SKM-1 cells

作　　者：杨碧慧 张玉琳[1] 王利[1] 罗小华[1] 陈礼平[1] 杨泽松[1] 刘林[1] 

机构地区：[1]重庆医科大学附属第一医院血液内科,重庆400016

出　　处：《第三军医大学学报》2015年第5期474-479,共6页Journal of Third Military Medical University

基　　金：重庆市自然科学基金重点项目(CSTC2013jj B0145)~~

摘　　要：目的构建SPAG6-shRNA慢病毒载体靶向沉默SPAG6基因,探讨SPAG6基因沉默对人骨髓增生异常综合征(myelodysplastic syndrome,MDS)细胞株SKM-1细胞增殖及凋亡的影响。方法设计并构建3条针对SPAG6基因的shRNA质粒表达载体,共转染293T细胞后得到可表达SPAG6基因RNAi的慢病毒载体。转染SKM-1细胞,利用流式细胞术检测转染效率,qRT-PCR及Western blot验证SPAG6基因干扰效果,筛选最佳靶点。CCK-8法检测SPAG6基因沉默对SKM-1细胞生长的影响,流式细胞术检测细胞凋亡。结果成功构建SPAG6-shRNA慢病毒载体,流式细胞术检测转染效率均在60%以上。qRT-PCR及Western blot检测结果显示SPAG6-shRNA3慢病毒载体敲除效率最高,为最佳靶点。SPAG6基因干扰后,干扰组SKM-1细胞的增殖明显受抑,抑制率为(60.18±0.37)%,与阴性对照组相比差别有统计学意义(P<0.05)。干扰组SKM-1细胞凋亡率为(16.32±5.80)%,显著高于阴性对照组[(4.28±0.88)%,P=0.024]及空白对照组[(3.08±1.50)%,P=0.019]。结论 SPAG6-shRNA慢病毒载体能有效降低SPAG6基因表达水平,抑制SKM-1细胞增殖并促进其凋亡。Objective To construct sperm-associated antigen 6（ SPAG6）-short hairpin RNA（ shRNA）-expressing lentiviral vector targeting SPAG6 gene,and investigate the effect of SPAG6 gene silencing on the proliferation and apoptosis of myelodysplastic syndrome（ MDS） cell line SKM-1. Methods Three kinds of shRNA-containing plasmids for interfering with SPAG6 gene expression were designed,constructed,and transfected into 293 T cells to obtain lentiviral vector expressing SPAG6-shRNA. Then,the lentiviral vector was transferred into SKM-1 cells, and the transfection efficiency was detected by flow cytometry. The interference efficiency of SPAG6 gene was determined by quantitative reverse transcription PCR and Western blotting,and the most effective shRNA-containing plasmid was screened. Cell proliferation was examined by CCK-8 assay,and cell apoptosis rate was detected by flow cytometry. Results SPAG6-shRNA-expressing lentiviral vector was successfully constructed,and the average transfection efficiency was higher than 60%. Quantitative reverse transcription PCR and Western blot results showed that SPAG6-shRNA3-expressing lentiviral vector was the most effective vector for knock-down of SPAG6 gene. After SPAG6 gene interference,the proliferation of SKM-1 cells in the interference group was inhibited significantly,and the inhibitory rate [（ 60. 18 ± 0. 37） %] was higher than that in the negative control group（ P 0. 05）.Meanwhile,the cell apoptosis rate of the interference group [（ 16. 32 ± 5. 80） %] was significantly higher than that of the negative control group [（ 4. 28 ± 0. 88） %,P = 0. 024] and that of the blank control group[（ 3. 08 ± 1. 50） %,P = 0. 019]. Conclusion SPAG6-shRNA-expressing lentiviral vector can effectively down-regulate the expression of SPAG6 gene,and significantly inhibit cell proliferation and promote apoptosis of SKM-1 cells.

关 键 词：SPAG6基因 慢病毒 RNA干扰 骨髓增殖异常综合征 

分 类 号：R394.33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]