CAJ | 학술논문

目的研究血管中骨髓间充质干细胞在不同钙化环境中的分化. 方法应用华法林,维生素K1及维生素D3制作血管钙化模型.造模成功后,分别取正常SD大鼠动脉和大鼠钙化动脉与骨髓间充质干细胞(MSC)共培养,分为3组:正常组:正常动脉+MSC;钙化诱导剂组:钙化诱导剂(地塞米松+β-甘油磷酸钠+ VitC)+正常动脉+MSC;钙化组:钙化动脉+MSC.各组均培养3周,实验第10天时采用酶联免疫吸附试验(ELISA)检测骨保护素(OPG)蛋白分泌情况,3周后倒置显微镜观察各组细胞形态变化,ELISA检测总蛋白含量和碱性磷酸酶(ALP)活性,反转录聚合酶链反应(RT-PCR)方法检测各组孤独受体2(Ror2) mRNA表达. 结果钙化组中MSC自发增殖向成骨样细胞分化.与正常组相比较,钙化组中总蛋白含量,骨代谢标志物ALP活性,OPG均明显升高,而Ror2 mRNA表达则显著低于正常组.钙化诱导剂组中MSC未向成骨样细胞分化,与正常组相比,ALP活性无明显差异,但总蛋白含量及OPG均升高,Ror2 mRNA表达则低于正常组.其中,钙化组Ror2 mRNA表达明显低于钙化诱导剂组. 结论在已钙化血管中,MSC向成骨样细胞转化,加重血管钙化;而当血管尚未发生钙化时,钙化诱导剂未诱导MSC向成骨样细胞分化,而是向平滑肌细胞分化,修复血管,这可能与血管中Ror2表达有关.
목적 연구혈관중골수간충질간세포재불동개화배경중적분화. 방법 응용화법림,유생소K1급유생소D3제작혈관개화모형.조모성공후,분별취정상SD대서동맥화대서개화동맥여골수간충질간세포(MSC)공배양,분위3조:정상조:정상동맥+MSC;개화유도제조:개화유도제(지새미송+β-감유린산납+ VitC)+정상동맥+MSC;개화조:개화동맥+MSC.각조균배양3주,실험제10천시채용매련면역흡부시험(ELISA)검측골보호소(OPG)단백분비정황,3주후도치현미경관찰각조세포형태변화,ELISA검측총단백함량화감성린산매(ALP)활성,반전록취합매련반응(RT-PCR)방법검측각조고독수체2(Ror2) mRNA표체. 결과 개화조중MSC자발증식향성골양세포분화.여정상조상비교,개화조중총단백함량,골대사표지물ALP활성,OPG균명현승고,이Ror2 mRNA표체칙현저저우정상조.개화유도제조중MSC미향성골양세포분화,여정상조상비,ALP활성무명현차이,단총단백함량급OPG균승고,Ror2 mRNA표체칙저우정상조.기중,개화조Ror2 mRNA표체명현저우개화유도제조. 결론 재이개화혈관중,MSC향성골양세포전화,가중혈관개화;이당혈관상미발생개화시,개화유도제미유도MSC향성골양세포분화,이시향평활기세포분화,수복혈관,저가능여혈관중Ror2표체유관.
Objective To study the differentiation of bone marrow mesenchymal stem cells in artery tissue under different calcification environments.Methods We made a vascular calcification model using warfarin,vitamin K1 and vitamin D3.After the model was successfully made,we took artery tissue of normal SD rat arteries and calcified arteries co-cultured with MSC,which were divided into three groups.The normal group included normal artery tissue with MSC; calcified inducers group included calcified inducers (dexamethasone with β-glycerophosphate and ascorbic acid),normal arterial tissue and MSC; calcification group included calcified artery tissue and MSC.Each group was cultured for 3 weeks.On the 10th day of the experiment,osteoprotegerin (OPG) protein secretion was detected by ELISA.After three weeks,changes of cell morphology were observed using inverted microscope,and total protein content and alkaline phosphatase (ALP) activity were detected by ELISA.In additional,the Ror2 (receptor tyrosine kinase-like orphan receptor 2) mRNA expression was detected by using RT-PCR method.Results MSC in calcification group spontaneously proliferated and differentiated to osteoblast-like cells.Compared with normal group,calcification group showed that the total protein content,ALP activity of bone metabolism markers,OPG were significantly elevated,while Ror2 mRNA expression was significantly decreased.MSCs in calcified inducers group did not differentiate to osteoblast-like cells,and the total protein content and OPG were increased,while ALP activity had no significant difference as compared with the normal group.However,Ror2 mRNA expression was lower in calcified inducers group than in normal group,while was higher than that in calcification group.Conclusions MSCs proliferate into bone-like differentiation in vascular calcification environment and aggravate the vascular calcification.And in normal vascular with calcified inducers environment,MSCs proliferate into smooth muscle cell differentiation and rehabilitate the vascular calcification.These phenomenons may be related to the Ror2 expression in artery.