原代大鼠肺动脉平滑肌细胞的提取和鉴定以及缺氧对其增殖的影响

目的提取SD大鼠肺动脉平滑肌细胞（PASMCs）并原代培养,为研究肺血管疾病提供体外模型,研究缺氧对大鼠PASMCs增殖的影响。方法组织贴壁法分离大鼠PASMCs,光镜观察细胞形态、透射电镜、α肌动蛋白（α-SM-actin）细胞免疫化学和细胞免疫荧光法进行鉴定。原代培养的PASMCs分别于常氧和/或低氧下培养2、6、12、24及48 h,用四甲基偶氮唑盐（MTT）比色法和增殖细胞核抗原（PCNA）细胞免疫化学法检测细胞增殖情况。结果光镜下PASMCs细胞为长梭形,呈典型的＂峰-谷＂状结构。免疫学检测显示胞浆被染成棕黄色,阳性率达96%以上。透射电镜下细胞呈长梭形,胞浆内有密斑、密体和大量肌丝,细胞器较少。MTT检测结果示各时间点缺氧组PASMCs吸光度（A值）均高于相对应的常氧组;与常氧组比较,低氧组A值在12 h时开始增加（P〈0.05）,24 h时增加最显著（P〈0.01）。与缺氧2 h组比较,A值在缺氧12 h开始增高（P〈0.05）,缺氧24 h增高最显著（P〈0.01）,而缺氧48 h与缺氧24 h基本一致。PCNA免疫学结果与MTT结果基本一致。结论用组织块贴壁法提取PASMCs,简单易行,且能得到纯度高、稳定生长的PASMCs。缺氧可以促进PASMCs的增殖。

Extraction and Identification of Primary Rat Pulmonary Artery Smooth Muscle Cells and Effects of Hypoxia on the Proliferation

Objective To extract and identify primary culture rat pulmonary arterial smooth cells(PASMCs),and investigate the effects of hypoxia on the proliferation of PASMCs.Methods Rat PASMCs were separated by the method of tissue block anchorage,and the cellular morphology was observed under light microscope.The cells were identified by projection electron microscopy,and α-smooth muscle actin(α-SM-actin) in the cells was identified by immunohistochemistry and immunofluorescence.The primary cultured PASMCs were exposed to normoxic and/or hypoxia condition for 2,6,12,24,48 hours respectively,then MTT assay and PCNA(proliferating cell nuclear antigen) immunohistochemistry were used to detect the proliferation of PASMCs.Results The cells tended to be long spindle and grew in the "peak-valley" mode under light microscope.Immunology results showed that endochylema was stained in brownish yellow,and the positive rate was beyond 96%.There were dense patch,dense body and many filaments in endochylema under projection electron microscopy.MTT assay demonstrated that the A values of PASMCs expose to hypoxia were higher than that of nomoxia.Comparing with normoxia,the A values of PASMCs exposed to hypoxia increased after 12 hours (P<0.05),significantly increased after 24 hours(P<0.01).Compared with 2 hours’ exposure to hypoxia,the A values increased after 12 hours(P<0.05),markedly increased after 24 hours(P<0.01),which after 48 hours was similar with 24 hours.The result of PCNA immunohistochemistry was consistent with that of MTT.Conclusions The tissue explants adherent method is simple and convenient,and can easily obtain rat PASMCs with high purity and stability.Hypoxia can promote the proliferation of PASMCs.