豚鼠脑基底动脉平滑肌细胞的原代培养及鉴定

Primary culture and identification of smooth muscle cells of cerebral basilar artery from guinea pigs

摘要:

目的 本研究联合使用直接贴壁法和酶消化法培养细胞，从豚鼠脑基底动脉中提取平滑肌细胞， 为相关实验研究提供材料。方法 从豚鼠颅内分离出脑基底动脉，用0.125% 胰蛋白酶进行消化并将组织块贴 壁，细胞用含20% 胎牛血清的DMEM/F-12 培养基进行培养。采用自然纯化法，并根据不同细胞贴壁能力的 差异性对细胞进行纯化。通过形态学观察、免疫荧光、免疫组织化学法及Western blot 检测对细胞进行鉴定。 结果 85% 组织块存活，第4 代平滑肌细胞纯度达95%。镜下可见细胞呈典型的“峰- 谷”样生长，免疫荧光、 免疫组织化学法及Western blot 检测显示，平滑肌细胞特异性的肌动蛋白呈阳性表达。结论 通过本实验可 以得到大量高纯度的豚鼠脑基底动脉平滑肌细胞，有望为颅内动脉粥样硬化等病理生理变化的体外研究及脑 血管平滑肌药物评价提供一个理想的实验材料。

Abstract:

Objective To obtain cerebral basilar artery smooth muscle cells from guinea pigs in combination with two common cultural methods, and provide materials for related experimental studies. Methods Cerebral basilar arteries were separated from posterior cranial fossa of guinea pigs first and enzymatically digested with 0.125% trypsin solution, allowed to adhere subsequently, the cells were cultured in DMEM/F-12 with 20% fetal calf serum, then purified based on the comprehensive application of natural purification and differential adherence ability. Finally, they were identified by morphological observation, immunofluorescence and immunocytochemistry analyses, as well as by Western blot. Results 85% of the explants survived. The smooth muscle cells at passage 4 had a purity of over 95%. The cells were observed as a typical "hill-and-valley" growth pattern under microscope. Moreover, immunofluorescent and immunohistochemical staining, and Western blot showed positive expression of α-SM-actin, a smooth muscle cell-specific marker. Conclusions By this protocol, a large number of highly purified cerebral basilar artery smooth muscle cells can be obtained from guinea pigs, which can provide an ideal experimental material for study of pathogenesis of the diseases related to intracranial atherosclerosis in vitro , as well as evaluation of drug effect on cerebral vascular smooth muscle.