CAJ | 학술논문

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可显著拮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蛋白表达上调(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基因沉默能拮抗谷氰酸对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.
목적 연구Par-4기인침묵대인골수간충질간세포(hBMSC)중Smac기인표체화반광안산단백수해매(Caspase)3매활성적영향급기항조망작용.방법 원대배양hBMSC.곡안산유도hBMSC조망.설계화합성량대침대Par-4기인mRNA적소RNA간우(siRNA)서렬(Par-4-SiRNA-1、2),구건진핵세포표체재체,용지질체개도전염hBMSC,이용G418사선은정표체세포주.실시형광정량PCR법검측Par-4 mRNA표체수평.류식세포술측정세포조망백분솔.Western인적측정Smac단백표체량.비색법측정Caspase-3매적활성.결과 설계적Par-4-SiRNA-1、2가현저유도hBMSC중Par-4기인침묵,Par-4 mRNA수평분별피강저88%、67%,곡안산유도hBMSC조망,조망백분솔위(58.9±8.9)%.Par-4-SiRNA-1가현저길항곡안산적유도조망작용,조망백분솔강위(37.2±6.3)%(F=58.26,P＜0.01).Par-4-SiRNA-1대곡안산유도적hBMSC중Smac단백표체상조(P＜0.01)화Caspase-3매활성상조(P＜0.01)유현저적억제작용(P＜0.01).결론 Par-4기인침묵능길항곡청산대hBMSC조망적유도작용.기궤제가능여Smac기인표체화Caspase-3매활성피억제유관.
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 0.12-4-0.03,0.33±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.