摘要
目的构建Hax1基因特异性小干扰RNA(sihax),检测sihax对Hax1基因的表达抑制,在293细胞中研究Hax1和Hax1siRNAs对细胞凋亡的影响。方法设计Hax1siRNA序列构建真核表达载体并转染到293细胞,倒置荧光显微镜下观察、流式细胞仪、AnnexinV-EGFP/PI双染法及RT-PCR检测靶基因Hax1的表达,以及293细胞的凋亡。结果成功构建了Hax1siRNA干扰质粒pBS/U6-sihaxA和pBS/U6-sihaxB,并且sihaxB对Hax1抑制效应明显强于sihaxA。过量表达Hax1可显著抑制293细胞的早期凋亡比例。用sihaxA和sihaxB抑制内源性Hax1表达,可使晚期细胞凋亡比例从对照的9.03%±0.473%分别增加到10.8%±0.513%(P<0.05)和16.6%±0.858%(P<0.01)。与此同时,sihaxB还可使早期细胞凋亡比例由37.3%±0.5%降低到32%±1.77%(P<0.01)。结论 Hax1siRNA对靶基因Hax1的抑制作用与靶序列的保守性有一定相关性。Hax1基因在细胞中参与凋亡相关的多种调节过程。本研究为进一步探讨Hax1功能奠定了基础。
Objective To construct the Hax1 gene specific siRNA（sihax） for detection of sihax mediated Hax1 gene repression and to study the influence of Hax1 and Hax1 siRNAs on cell apoptosis.Methods The oligo sequences of Hax1 siRNAs were selected,and the constructed eukaryotic expression vectors were transfected into 293 cells.The influence of Hax1 and its siRNAs on cell apoptosis was evaluated by a number of approaches such as inverted fluorescence microscope,flow cytometric analysis,annexin V-EGFP/PI double staining,and standard RT-PCR.Results Hax1 specific siRNAs,pBS/U6-sihaxA and pBS/U6-sihaxB,were successfully constructed.Analysis by RT-PCR and inverted fluorescence microscopy demonstrated that the inhibitory effect induced by sihaxB was stronger than that of sihaxA.Over-expression of Hax1 significantly inhibited the early apoptosis of 293 cells.Inhibiting the endogenous Hax1 gene expression by by sihaxA and sihaxB markedly increased late apoptotic cells from 9.03%±0.473% to 10.8%±0.513%（P0.05） and 16.6%±0.858%（P0.01）,respectively.In the same time,sihaxB also reduced the early apoptotic cells from 37.3%±0.5% to 32%±1.77%（P0.01）.ConclusionThe sequence conser vation of the targeting site may influence the inhibitory effect induced by Hax1 siRNA.Hax1 gene might actively involve in the multiple regulatory processes related to cell apoptosis.This study provides some valuable information for the functional study on Hax1 gene in the future.
出处
《基础医学与临床》
CSCD
北大核心
2011年第6期602-608,共7页
Basic and Clinical Medicine
基金
国家自然科学基金(30871283)
2010年中国医学科学院基础医学研究所所院长基金(2010PYZ17)
