大鼠平滑肌细胞和平滑肌祖细胞的体外培养及二者间表型转化的初步研究  

In vitro culture of vascular smooth muscle cells and smooth muscle progenitor cells from rat and preliminary study of phenotype transforming between two cells

作　　者：谢晓晨[1] 李飞[1] 包楠迪[1] 左惠荣[1] 张荣庆[1] 郭文怡[1] 

机构地区：[1]解放军第四军医大学西京医院心内科,西安710032

出　　处：《中华保健医学杂志》2013年第5期411-414,共4页Chinese Journal of Health Care and Medicine

基　　金：国家自然科学基金面上项目(81170183)

摘　　要：目的探讨体外分离培养大鼠血管平滑肌细胞(VSMC)和平滑肌祖细胞(SPC)的方法,并初步比较不同培养条件下大鼠SPC对VSMC表型转化的影响。方法原代培养细胞,通过蛋白印迹实验(Western Blot)检测平滑肌α-肌动蛋白(α-SMA)鉴定VSMC,SPC培养后经流式细胞术鉴定分选贴壁细胞CD14和CD105双阳性表达细胞继续培养为SPC,通过SPC与VSMC共培养以及利用Transwell装置条件培养、空白对照3种条件干预后,应用Western Blot法分别测定三组骨桥蛋白水平变化,以检测并初步比较不同条件下SPC对VSMC表型转化的影响。结果两种细胞分别原代培养后,Western Blot鉴定VSMC,结果显示α-SMA表达呈阳性;鉴定SPC免疫细胞化学染色α-SMA表达呈阳性,且流式细胞仪分析显示CD14和CD105呈双阳性表达。与空白对照相比,两种细胞共同培养和条件培养时VSMC表达骨桥蛋白水平明显升高,共同培养较条件培养骨桥蛋白水平升高效果更为明显。结论 SPC对VSMC的表型由收缩型向合成型转变具有一定作用,SPC通过直接作用与旁分泌作用不同程度地增强了VSMC的表型转化;对比发现,两细胞直接接触培养时VSMC表型转化的影响更为明显(P<0.05)。Objective To investigate the method of in vitro culturing VSMC and SPC,and to observe the phenotype transforming between these two cells under different cultivating condition. Methods Primary cells culture, Detect a-smooth muscle actin（α-SMA） antibody by Western Blot to identify VSMC, screen SPC which are positive for both CD14 and CD105 by flow cytometry and then keep on cultivating them. Co-culture SPC with VSMC and interfere VSMC with SPC in conditioned medium. Determine the expression level of osteopontin （OPN） to observe the influence of SPC to the phenotype transforming of VSMC. Results The detection of VSMC expression of α-SMA antibody by Western blot turned to be positive, results of immuncytochemistry of SPC was positive and SPC was positive for both CD14 and CD105. Compared with control group,the expression level of OPN rise obviously in both co- culture group and transwell group,especially in co-culture group （P〈0.05）. Conclusions SPC plays a certain role in the process of phenotype transforming by direct contact effection and paracrine effection, and the effection of direct contact is more obvious.

关 键 词：血管平滑肌细胞 平滑肌祖细胞 细胞培养 表型转化 

分 类 号：R33-33[医药卫生—人体生理学]