摘要
目的研究Par-4基因沉默对人骨髓间充质干细胞（hBMSC）中Smac基因表达和半胱氨酸蛋白水解酶（Caspase）3酶活性的影响及其抗凋亡作用。方法原代培养hBMSC。谷氨酸诱导hBMSC凋亡。设计和合成两对针对Par-4基因mRNA的小RNA干扰（siRNA）序列（Par-4-SiRNA-1、2），构建真核细胞表达载体，用脂质体介导转染hBMSC，利用G418筛选稳定表达细胞株。实时荧光定量PCR法检测Par-4 mRNA表达水平。流式细胞术测定细胞凋亡百分率。Western印迹测定Smac蛋白表达量。比色法测定Caspase-3酶的活性。结果设计的Par-4-SiRNA-1、2可显著诱导hBMSC中Par-4基因沉默，Par-4 mRNA水平分别被降低88％、67％，谷氨酸诱导hBMSC凋亡，凋亡百分率为（58．9±8．9）％。Par-4-SiRNA-1可显著拈抗谷氨酸的诱导凋亡作用，凋亡百分率降为（37．2±6．3）％（F=58．26，P〈0．01）。Par-4-SiRNA-1对谷氨酸诱导的hBMSC中Smac蛋白表达上凋（P〈0．01）和Caspase-3酶活性上调（P〈0．01）有显著的抑制作用（P〈0．01）。结论Par-4基因沉默能拈抗谷氨酸对hBMSC凋亡的诱导作用。其机制可能与Smac基因表达和Caspase-3酶活性被抑制有关。
Objective To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells （hBMSCs）. Methods Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs （Par-4-siRNA-1 and -2 ） targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups： non-transfected hBMSCs （normal control group）, blank Pae-4 plasmid transfected hBMSCs （Par-4 control group）, Par-4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups： non-transfected normal hBMSCs, glutamate （an apoptosis inducer） ＋ non-transfected hBMSC group, glutamine ＋ Par-4-siRNA-1 hBMSC group, and glutamate ＋ Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression. Results The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SIENA-2 hBMSCs, and Par-4 control hBMSCs were 0. 12 ± 0. 03, 0.33 ± 0.09, and 0.97 ±0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate ＋ Par-4-siRNA-1 hBMSCs was （ 37.2 ± 6.3 ） %, significantly lower than that of the glutamate ＋ non-transfected hBMSC group [ （58.9 ± 8.9） % , F = 58.26, P 〈 0.01 ）. The Smac protein expression level of the glutamate ＋ non-transfected hBMSC group was significantly higher than that of the normal control group （ P 〈0.01 ） ; however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate ＋ non-transfected hBMSC group （ P 〈 0.01 ）. The caspase-3 activity of the glutamate ＋ Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate ＋ non-transfected BMSC group （ P 〈 0. 01 ）. Conclusion Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第6期411-415,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目（30570863）
江苏省卫生厅医学科技发展重大基金资助项目（K200504）
