短发夹RNA缄默survivin基因对Jurkat细胞增殖及凋亡的影响

Effects of Short Hairpin RNA-Inhibited Survivin Gene on Proliferation and Apoptosis of Jurkat Cell

目的探讨survivin短发夹RNA(shRNA)表达质粒对白血病细胞株体外生长和凋亡的影响。方法酶切法构建干扰survivin基因表达的重组质粒和阴性对照质粒,电击转染质粒入Jurkat细胞。实验分为未转染组、无功能shRNA组(阴性对照组)及重组质粒组。分别采用反转录聚合酶链反应(RT-PCR)及免疫组织细胞化学染色法检测各组细胞中survivin mRNA、蛋白的表达,四甲基偶氮唑蓝(MTT)比色法测定各组细胞的生长抑制率,流式细胞仪计数(FCM)测定各组细胞凋亡率。结果DNA测序证实sur-vivin短发夹RNA表达质粒和阴性对照质粒构建成功。重组质粒组与未转染组及无功能shRNA组比较,RT-PCR结果显示转染后重组质粒转染组survivin基因表达相对于未转染组及阴性对照组显著下降(Pa<0.05);免疫组织细胞化学染色结果显示重组质粒转染组survivin蛋白表达相对于未转染组及阴性对照组显著下降;MTT显示重组质粒生长抑制率为(24.65±1.10)%,而未处理组为(2.04±0.16)%,阴性质粒组为(2.17±0.15)%;FCM结果示未转染组细胞凋亡率为(2.13±0.42)%,阴性质粒组凋亡率为(2.55±0.95)%,重组质粒转染组细胞凋亡率为(11.48±1.11)%。shRNA重组质粒介导的RNA干扰抑制survivin表达,抑制Jurkat细胞生长和促进其凋亡(P<0.05)。结论靶向为survivin基因的RNA干扰技术可为儿童白血病治疗提供新的手段。

Objective To explore the influence of short hairpin RNA （shRNA） targeting survivin gene on proliferation and apoptosis in human leukemia cell line in vitro. Methods shRNA - survivin expression vector and negative expression vector was constructed and transfected into Jurkat cells by electroporation. The experiment included 3 groups：untransfected group, negative plasmid group and recombinant plasmid group. Reverse transcription PCR （RT -PCR） was performed to analyze the expression of survivin mRNA. Survivin protein was examined by immunohistochemistry. Methyl thiazolyl tetrazolium（MTT） assay was used to analyze the proliferation of cells. Flow cytometry （FCM） was used to detect the apoptosis of cells. Results DNA sequencing confirmed the recombinant plasmid and the negative plasmid was successfully constructed. Compared with untransfected group and negative plasmid group,the expression of survivin mRNA decreased in recombinant plasmid group （ Pa 〈 0.05 ） ; immunohistochemistry results showed that the survivin protein expression decreased in recombinant plasmid group, compared with untransfected group and negative plasmid group ; MTF assay revealed the growth inhibition ratio of the untransfected group was （2.04 ± 0.16 ）% , the growth inhibition ratio of negative plasmid group was （2.17 ± 0.15 ）% , the growth inhibition ratio of recombinant plasmid group was （24.65 ± 1.10 ） % ; the cell apoptotic rate of recombinant plasmid was （ 11.48 ± 1.11 ） % by FCM, the cell apoptotic rate of the untransfected group was （2.13 ±0.42） % ,and the Cell apoptotic rate of the negative plasmid group was （2.55 ±0.95） %. Recombinant plasmid could effectively reduce the expression of survivin gene, inhibit growth of transfected Jurkat cells and enhance cell apoptosis （P 〈 0.05 ）. Conclusion RNA interference targeting survivin gene provides a new approach to children＇s leukemia.