大鼠脑动脉平滑肌细胞的体外培养与鉴定

In vitro culture and identification of smooth muscle cells from rat intracranial cerebral artery

目的建立大鼠颅内脑动脉平滑肌细胞(smooth muscle cells,SMCs)的体外培养方法。方法无菌条件下分离大鼠脑基底动脉,去除血管外膜后剪成约0.2mm的小段,分别用0.1% Ⅰ型胶原酶、0.125%胰蛋白酶消化,细胞用含20%胎牛血清的DMEM/F12培养基进行培养。采用人工刮除法及差速贴壁法纯化细胞。SMCs通过形态学观察及α-平滑肌肌动蛋白免疫细胞化学方法鉴定。结果原代培养3d后细胞开始贴壁,2w后细胞呈梭形,汇合后具有典型的"峰-谷"生长特点。传代培养的细胞保持上述特征,第5代细胞经α-平滑肌肌动蛋白表达鉴定,纯度达97%以上。台盼蓝排斥实验检测细胞存活率>95%。结论这种方法操作简单、结果可靠、成本低廉,可为颅内动脉粥样硬化等脑血管病机制和治疗的研究提供适宜的体外培养细胞模型。

Objective To develop a method of culturing smooth muscle cells （SMCs） derived from rat intracranial cerebral artery in vitro. Methods Rat basilar arteries were isolated under sterile conditions. After removal of the adventitia,they were cut into approximately 0.2mm rings and then digested with 0.1% typeⅠcollagenase and with 0.125% trypsin,respectively. After digestion,cells were cultured in DMEM/F12 with 20% fetal calf serum. The cells were purified by using a combination of manual scraping and differential attachment techniques. SMCs were identified by the morphological criteria and immunocytochemical staining of smooth muscle α-actin. Results After 3 days of incubation,primary cultures of cells began to attach to the wall of the incubation dishes. After 2 weeks,cells were exhibited a spindle-shaped morphology with a classic ＂hill-and-valley＂ growth pattern at confluence. Those features remained unchanged throuh subsequent passages. The purity of fifth passaged SMCs was greater than 97% as confirmed by their expression of smooth muscle α-actin. Trypan blue exclusion assays demonstrated95% cell viability. Conclusion The method described here is a relatively simple,reliable and inexpensive for establishing an in vitro cell culture model,which is suitable for studying the mechanism and treatment of cerebrovascular diseases such as intracranial atherosclerosis.