CAJ | 학술논문

目的探讨小RNA干扰靶向抑制Par-4基因表达对人骨髓间充质干细胞凋亡的影响.方法原代培养人骨髓间充质干细胞,谷氨酸诱导凋亡.设计和合成针对Par-4基因mRNA的siRNA(Par-4-siRNA),构建真核细胞表达载体,用脂质体介导转染入骨髓间充质干细胞,利用G418筛选稳定表达细胞株.实时荧光定量PCR(real-time PCR)法检测Par-4 mRNA的表达量.流式细胞术测定细胞凋亡百分率.Western blot测定磷酸化(Thr308)的蛋白激酶Akt1蛋白表达量.比色法测定Caspase-3酶活性.结果筛选出的Par-4-SiRNA-1、2显著抑制人骨髓间充质干细胞中Par-4基因的mRNA表达,mRNA水平分别降低88%、67%.在谷氨酸诱导后24 h,人骨髓间充质干细胞发生凋亡,百分率为60.30%±6.82%.Par-4-SiRNA-1、2都显著抑制谷氨酸的诱导作用,凋亡百分率降为38.80%±3.97%(P＜0.01)和45.49%±4.32%(P＜0.01).谷氨酸诱导后24 h人骨髓问充质干细胞中磷酸化Akt1蛋白表达下调(89.07±6.42和28.30±5.65,P＜0.01),而Par-4-SiRNA-1、2对之下调都有显著抑制作用(63.56±6.75和45.59±4.88,均有P＜0.01).谷氨酸诱导后24 h人骨髓间充质干细胞中Caspase-3酶活性上调(0.1428±0.0495和0.8616±0.1051,P＜0.01),而Par-4-SiRNA-1、2对之上调都有显著抑制作用(0.6581±0.0555和0.7041±0.0401,均有P＜0.01).结论小RNA干扰可靶向抑制Par-4基因的表达,拮抗谷氨酸诱导的人骨髓间充质干细胞的凋亡.
목적 탐토소RNA간우파향억제Par-4기인표체대인골수간충질간세포조망적영향.방법 원대배양인골수간충질간세포,곡안산유도조망.설계화합성침대Par-4기인mRNA적siRNA(Par-4-siRNA),구건진핵세포표체재체,용지질체개도전염입골수간충질간세포,이용G418사선은정표체세포주.실시형광정량PCR(real-time PCR)법검측Par-4 mRNA적표체량.류식세포술측정세포조망백분솔.Western blot측정린산화(Thr308)적단백격매Akt1단백표체량.비색법측정Caspase-3매활성.결과사선출적Par-4-SiRNA-1、2현저억제인골수간충질간세포중Par-4기인적mRNA표체,mRNA수평분별강저88%、67%.재곡안산유도후24 h,인골수간충질간세포발생조망,백분솔위60.30%±6.82%.Par-4-SiRNA-1、2도현저억제곡안산적유도작용,조망백분솔강위38.80%±3.97%(P＜0.01)화45.49%±4.32%(P＜0.01).곡안산유도후24 h인골수문충질간세포중린산화Akt1단백표체하조(89.07±6.42화28.30±5.65,P＜0.01),이Par-4-SiRNA-1、2대지하조도유현저억제작용(63.56±6.75화45.59±4.88,균유P＜0.01).곡안산유도후24 h인골수간충질간세포중Caspase-3매활성상조(0.1428±0.0495화0.8616±0.1051,P＜0.01),이Par-4-SiRNA-1、2대지상조도유현저억제작용(0.6581±0.0555화0.7041±0.0401,균유P＜0.01).결론 소RNA간우가파향억제Par-4기인적표체,길항곡안산유도적인골수간충질간세포적조망.
Objective The prostate apoptosis response factor-4 (Par-4) gene was originallyidentified by differential screening for genes that are up-regulated when prostate cells are induced to undergoapoptosis. Par-4 was found to possess potent apoptotic activity in various cellular systems in response tonumerous stimuli. The aim of this study was to explore the effects of small interfering RNA (siRNA) againstPar-4 gene on the apoptosis of human bone marrow mesenchymal stem cells (hBMSCs) exposed toglutamate. Methods Primary culture of hBMSCs was carried out and siRNAs targeted Par-4 gene ( Par-4-SiRNA) were chemically synthesized. Eukaryocytic expression vector was built and were transfeeted intohBMSCs with liposome. After selecting with G418, the stable cell clones were treated with glutamate. Theexpression of Par-4 mRNA was determined by real-time PCR. The apoptosis of hBMSCs was quantified byflow cytometry. Western blotting was used to detect the protein levels of phosphorylated Aktl ( Thr308 ).Relative Caspase-3 activity was determined by eolorimetric assay. Results The Par-4-SiRNA-1 and Par-4-siRNA-2 could markedly down-regulate the mRNA levels of Par-4 gene in hBMSCs. With the transfeetious ofPar-4-SiRNA-1 and Par-4-SiRNA-2, the levels of Par-4 mRNA were respectively decreased by 88% and67%. Both Par-4-SiRNA-1 and Par-4-SiRNA-2 inhibited significantly the apoptosis of hBMSCs induced byglutamate, in which the percentages of apeptotic cells were respectively decreased to 38. 80% 4- 3.97% ( P＜0. 01 ) and 45.49% + 4. 32% ( P＜0.01 ) from 60. 30% 4- 6. 82%. Western blot assays demonstrated that,glutamate dewn-regulated the expression of phaspborylated Aktl proteins in hBMSCs (89. 07±6. 42 and28.30±5.65, respectively, P＜0. 01 ). However,Par-4-SiRNA-1 and Per-4-SiRNA-2 could markedlyrecover the down-regulatiun of Akt1 proteins induced by glutamate (63. 56±6. 75 and 45.59±4. 88,respectively, P＜0. 01 ). And the relative Caspase-3 activity which was enhanced by the treatment withglutamate(0. 1428±0. 0495 and 0. 8616±0. 1051, P＜0. 01 ), was suppressed by Par-4-SiBNA-1 and Par-4-SiBNA-2(0.8616±0. 1051 and0.6581±0.0555, respectively, P＜0.01). Conclusion SiRNA againstPar-4 gene could inhibit the apoptosis of hBMSCs induced by glutamate, and its inhibitory effects may bemediated by the up-regulation of phosphorylated Akt1 and the suppression of the relative Caspase-3 activity.