冷酶消化法分离和培养新生大鼠许旺细胞

背景：常规酶消化法在许旺细胞分离培养实验中应用较多,但由于胰蛋白酶在37 ℃超过30 min对细胞有毒性作用,会对许旺细胞的数量和纯度有一定的影响。 目的：探索快速分离和培养大量许旺细胞的新方法。 方法：采用4 ℃胰蛋白酶消化剥离好的乳鼠坐骨神经6~18 h分离许旺细胞,培养一段时间,进行MTT实验绘制细胞生长曲线和苏木精-伊红染色和免疫组织化学染色观察许旺细胞的形态,并对阳性细胞进行计数。 结果与结论：结果得到的细胞数量超过1×10⁶,经抗S-100蛋白免疫组织化学染色对细胞进行鉴定,细胞纯度达到90%以上。从培养的细胞形态和细胞生长曲线可见,冷酶消化法是一种简单快捷的方法,其细胞具有良好的生物学特性,在分离时间、细胞数量和纯度、细胞的生物活性均达到临床上的要求。

Application of cold enzyme digestion in isolating and culturing Schwann cells from newborn rats

BACKGROUND:Currently, conventional enzymatic digestion methods are commonly used to isolate and culture Schwann cells. Because trypsinase has toxic effects on Schwann cells in 30 minutes at 37 ℃, the amount and purity of Schwann cells can have a certain impact. OBJECTIVE:To improve a novel method to rapidly isolate and culture a great amount of Schwann cells. METHODS:Schwann cells were isolated from newborn rat sciatic nerve digested with trypsinase at 4 ℃, 6 to 18 hours, and then cultured for some time. MTT was used to draw cell growth curve. Hematoxylin-eosin staining and immunohistochemistry were used to observe cell morphology, and positive cells were counted. RESULTS AND CONCLUSION:The quantity of Schwann cells was over 1×106 and the purity of Schwann cells was over 90% assessed by anti-s100 protein immunohistochemistry stainer. From the morphology of cultured cells and cells growth curve, the cold enzyme digestion method was simply and effective, the purified cells had good biologic characteristics. In conclusion, the novel method can satisfy the clinical need such as isolating and culturing time, the quantity and purity of cells and the bioactivity of Schwann cells.