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目的探讨采用酶消化法快速分离SD大鼠胸、腹主动脉平滑肌细胞的方法,为膜片钳实验提供大量原代平滑肌细胞。方法取SD大鼠胸、腹主动脉,用酶液Ⅰ(木瓜蛋白酶等)、酶液Ⅱ(胶原酶和透明质酸酶等)酶解分离平滑肌细胞,酶液Ⅰ、Ⅱ的消化时间分别选取10、15、20、25、30 min,并两两组合以确定最佳消化时间。分离的细胞经台盼蓝染色测定其活性并通过平滑肌特异性α-肌动蛋白(α-actin)免疫组化染色鉴定。结果分离的平滑肌细胞在倒置显微镜下计数,酶液Ⅰ、Ⅱ的消化时间分别在30、15 min时平滑肌细胞数量最多为(18.3±1.5)个/低倍视野,与其他各时间点组合相比,差异有统计学意义(P<0.05)。镜下观察典型的平滑肌细胞呈长梭形,细胞膜完整,边缘光滑;台盼蓝染色,90%以上的细胞为活细胞;免疫组化染色鉴定为平滑肌细胞。结论酶消化法分离获取SD大鼠平滑肌细胞方法简单、成本低,采用本法可获得较大量的平滑肌细胞供实验使用。
OBJECTIVE: To explore a method for rapid isolation of smooth muscle cells from thoracic and abdominal aorta of SD rats by enzymatic digestion and to provide a large number of primary smooth muscle cells for patch clamp experiments. Methods Thoracic and abdominal aorta were isolated from SD rats, and the smooth muscle cells were separated by enzymolysis Ⅰ (papain and so on) and enzyme Ⅱ (collagenase and hyaluronidase) 10,15,20,25,30 min, and every two combinations to determine the optimal digestion time. Isolated cells were assayed for trypan blue staining and identified by smooth muscle specific alpha-actin immunohistochemical staining. Results The isolated smooth muscle cells were counted under an inverted microscope. The digestive time of enzyme Ⅰ and Ⅱ were (18.3 ± 1.5) / low magnification at 30 and 15 min, respectively. Compared with other time points , The difference was statistically significant (P <0.05). Microscopic observation showed that the typical smooth muscle cells were long fusiform, cell membrane integrity, smooth edges; trypan blue staining, more than 90% of cells were viable cells; immunohistochemical staining of smooth muscle cells. Conclusion Enzymatic digestion method to obtain SD rat smooth muscle cells is simple, low cost, using this method can obtain a larger number of smooth muscle cells for experimental use.