短发夹RNA诱导survivin基因缄默对Jurkat细胞凋亡和增殖的影响  

Effect of shRNA-mediated survivin gene silencing on apoptosis and proliferation of leukemia cell line Jurkat

作　　者：古聪敏[1] 朱有凯[2] 吴红阳[1] 张萌[1] 廖冰[3] 林汉良[3] 

机构地区：[1]中山大学第一附属医院病理科 [2]广西壮族自治区人民医院病理科 [3]中山大学第一附属医院病理科广州510080

出　　处：《中华血液学杂志》2006年第6期394-397,共4页Chinese Journal of Hematology

摘　　要：目的研究应用载体表达短发夹 RNA(short hairpin RNA,shRNA)干扰技术抑制人 T 淋巴细胞白血病细胞株 Jurkat 细胞 survivin 基因的表达,探讨 survivin 基因表达缄默对 Jurkat 细胞凋亡和增殖的影响。方法构建针对 survivin 基因的 shRNA 重组质粒并转染至 Jurkat 细胞,分别用多重 PCR和 Western blot 法检测瞬时转染和稳定转染细胞 survivin 基因 mRNA 和蛋白表达水平的变化;流式细胞术检测瞬时转染和稳定转染细胞凋亡指数的变化,绘制细胞生长曲线,探讨 survivin shRNA 转染对细胞生长和增殖的影响。结果多重 PCR 结果示与无功能(对照组)shRNA 处理组和磷酸盐缓冲液处理组比较,survivin shRNA 瞬时转染和稳定转染细胞 surivivin 基因 mRNA 表达均显著下降,抑制率分别为66.675% 和60.69%(P<0.05);Western blot 结果显示 survivin shRNA 瞬时转染和稳定转染细胞survivin 蛋白表达水平亦显著降低,抑制率分别为63.41% 和60.18%(P<0.05)。流式细胞术检测瞬时转染和稳定转染细胞的凋亡率显著增加,分别为(22.41±2.83)% 和(20.73±2.56)%(与对照组比较,P均<0.05),细胞倍增时间显著延长。生长曲线显示稳定转染细胞的生长显著减慢。结论shRNA 重组质粒介导的 RNA 干扰能明显抑制 Jurkat 细胞 survivin 基因 mRNA 和蛋白产物的表达,诱导细胞凋亡和生长抑制。Objective To transfect a short hairpin RNA （shRNA） against survivin gene into human T lymphoblastic leukemia cell line Jurkat, and to explore the effects on apoptosis and proliferation of transfected cells. Methods The survivin-shRNA expression vector were constructed and transfeeted into Jnrkat cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blot analysis respectively. Apoptosis index of transfected Jurkat cells was quantified by flow eytometry. The potential of cell proliferation was described by cell growth curves. Results In survivin-shRNA Iransfected Jurkat cells, survivin mRNA levels were significantly redt,ced by 66.67% （transient transtfetion） and 60.69% （stable transfection） respectively, compared with that in eontrol-shRNA treated group and PBS treated group （P 〈0.05）; and the levels of survivin protein were significantly reduced by 63.41% （transient transfection） and 60.18% （stable transfection）, compared wilt, that it, the two control groups （P 〈 0.05）. Apoptosis index was significantly increased during both transient and stable transfection, respectively [ （22. 41 ± 2. 83 ） % and （20.73 ±2.56） % （P 〈0.05）]. Survivin-shRNA also inhibited the proliferation of Jurkat cells. Conclusions Vector-based survivin-shRNA can effectively reduce the expression of survivin gene, induce apoptosis and inhibit growth of transfected Jurkat cells.

关 键 词：基因 survivin RNA干扰 细胞凋亡 细胞系 Jurkat 

分 类 号：R346[医药卫生—基础医学]