小鼠主动脉血管平滑肌细胞分离培养及鉴定  

Isolated culture and identification of mouse aortic vascular smooth muscle cell

作　　者：辛毅[1,2] 张耀中[1,3] 林筝[1,2] 张颖[1,2] 秦彦文[1,3] 

机构地区：[1]首都医科大学附属北京安贞医院 [2]北京市心肺血管疾病研究所实验中心,北京100029 [3]北京市心肺血管疾病研究所血管生物研究室,北京100029

出　　处：《新乡医学院学报》2012年第1期5-10,共6页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:81070251;81100222);省部共建教育部心血管重塑相关疾病重点实验室项目(编号:110267)

摘　　要：目的建立体外分离培养小鼠主动脉血管平滑肌细胞(VSMC)的技术方法并观察其生长特征。方法比较组织贴块法及酶联合消化法原代分离小鼠主动脉来源的VSMC,利用倒置显微镜观察细胞生长情况;苏木精-伊红(HE)染色观察细胞形态及免疫荧光染色法鉴定细胞;台盼蓝法、绘制生长曲线法、3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法分别测定主动脉VSMC传代细胞的成活率、生长、增殖特征;观察不同血清含量对主动脉VSMC生长的影响。结果组织贴块法培养的细胞6 d后,细胞从组织块边缘长出,14 d后生长迅速可传代;酶联合消化法分离培养的细胞3 d后,细胞呈梭形生长,7～8 d后生长迅速可传代;第2代细胞在显微镜下观察可见呈典型"峰-谷"状生长;2种方法获得的细胞行免疫荧光染色显示胞浆内α-平滑肌肌动蛋白表达阳性,细胞成活率均为96%;细胞生长曲线近似"S"形,MTT法显示细胞生长第3～5天光密度值变化较明显;用含体积分数20%血清的杜尔伯科改良伊格尔培养基高糖培养液培养的细胞出现明显对数期。结论本研究建立了高效分离和培养主动脉VSMC的2种方法,细胞均稳定地表达α-平滑肌肌动蛋白,为血管疾病的研究提供了理想的细胞模型。Objective To build the technical method of isolated culture mouse aorta vascular smooth muscle cell （VSMC） in vitro and observe their growth characteristics. Methods VSMC of original generation separation from aorta in mice by tissue piece inoculation and enzymatic digestion method were compared and the conditions of cellular growth were ob- served under inverted microscope. The morphology of cell were observed with hematoxylin-eosin （ HE ） staining and identified by i staining. The survival ratio, growth and proliferation characteristics of passage VSMC were measured by trypan blue method, growth curve method, 3- （ 4,5-dimethyl-2 -thiahiazo ） -2,5-diphenyltetrazolium bromide （ MTT ）. The effects of different serum content on growth of aorta VSMC were observed. Results After cells were cultured for six days with tissue piece inoculation method, VSMC were slowly grown from the tissue edge and rapidly growth until 14 days and could be passaged. After cells were cultured for three days with enzyme digestion method, the cells were spindle growth and rapidly growth until 7 - 8 days and could be passaged. The second generation cells had a typical ＂peak-valley＂ like growth under microscope. The expressions of intracellular a-smooth muscle actin in cells obtained by two methods were positive by immunofluorescence stain,cell survival rate was 96%. Cells growth curve were similar to ＂S＂ shape,MTT assay showed that optical density change of cell growth was obvious at 3 - 5 days. Containing 20% （ V ： V） serum DMEM high glucose culture medium of cells had an affected on apparent logarithmic phase. Conclusion This study provided two efficient isolation methods to culture mouse aortic VSMC, which stably express a-smooth muscle actin. It may be provide an ideal cellular model for the research of vascular diseases.

关 键 词：小鼠 血管平滑肌细胞 原代培养 鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]