CAJ | 학술논문

目的构建myocardin特异性siRNA真核表达载体,体外观察其对myocardin基因的沉默效应及对小鼠骨髓间充质干细胞(MSC)向平滑肌样细胞分化过程中的影响.方法培养小鼠骨髓问充质干细胞并利用血小板源生长因子(PDGF-BB,50 mg/L)联合高浓度胎牛血清(20%FBS)诱导其向平滑肌样细胞分化;构建含U6启动子和myocardin特异短发卡RNA(shRNA)编码序列的质粒载体pGen-myo-shRNA,以含非特异性shRNA编码序列的质粒载体pGenesil-Con(pCon)为阴性对照,分别转染诱导培养后第6天的小鼠MSC,48 h后用RT-PCR技术检测细胞内myocardin在mRNA水平的表达;同时用免疫组织化学方法检测平滑肌肌球蛋白重链(sM myosin heavy chain,SM-MHC)的表达来鉴定平滑肌样细胞的分化.结果成功构建myocardin特异性siRNA真核表达载体,利用其沉默myocardin基因后,干扰组相比空白对照组myocardin mRNA表达下调42.86%,差异有统计学意义(P<O.01);且免疫组织化学结果表明:干扰组SM-MHC阳性表达低于空白对照组(P<0.01).结论小鼠骨髓间充质干细胞中存在少数细胞具有分化成为平滑肌样细胞的潜能;构建的pGen-myo-shRNA重组质粒能有效抑制myocardin基因的表达,且对小鼠骨髓问充质干细胞向平滑肌样细胞分化有抑制作用.
목적 구건myocardin특이성siRNA진핵표체재체,체외관찰기대myocardin기인적침묵효응급대소서골수간충질간세포(MSC)향평활기양세포분화과정중적영향.방법 배양소서골수문충질간세포병이용혈소판원생장인자(PDGF-BB,50 mg/L)연합고농도태우혈청(20%FBS)유도기향평활기양세포분화;구건함U6계동자화myocardin특이단발잡RNA(shRNA)편마서렬적질립재체pGen-myo-shRNA,이함비특이성shRNA편마서렬적질립재체pGenesil-Con(pCon)위음성대조,분별전염유도배양후제6천적소서MSC,48 h후용RT-PCR기술검측세포내myocardin재mRNA수평적표체;동시용면역조직화학방법 검측평활기기구단백중련(sM myosin heavy chain,SM-MHC)적표체래감정평활기양세포적분화.결과 성공구건myocardin특이성siRNA진핵표체재체,이용기침묵myocardin기인후,간우조상비공백대조조myocardin mRNA표체하조42.86%,차이유통계학의의(P<O.01);차면역조직화학결과 표명:간우조SM-MHC양성표체저우공백대조조(P<0.01).결론 소서골수간충질간세포중존재소수세포구유분화성위평활기양세포적잠능;구건적pGen-myo-shRNA중조질립능유효억제myocardin기인적표체,차대소서골수문충질간세포향평활기양세포분화유억제작용.
Objective Construction of a sinall interfering RNA(siRNA)eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesonchymal stem cells(MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro. Methods Mouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum(20%).Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector. which contained U6 promoter.The recombinant plasmid and control plasmid were transfeeted into MSCs which had been cultured with PDGF-BB for 6 days beforehand.The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection.Immunohistochemistry Was used to detect the SM-MHC and tO identify the smooth muscle-like cells.Results The recombinant plasmids carrying myocardin-siRNA sequences were constructed sueeessfully and the myocardin mRNA was reduced 42.86% by pGen-myo- shRNA in comparing with that of the controls(P<0.01);and the expression of SM-MHC protein was down- regulated(P<0.01).Conclusion Subset of mouse MSCs have the potential to differentiate into smooth muscle-like cells,a possible cell source responsible for atheroselerotic plaque formation.and myocardin expression may play an important role during this process.