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背景:许旺细胞移植可改变损伤局部的微环境,有利于神经创伤的修复,获取大量高纯度、具有增殖活性的许旺细胞是研究的关键。目的:探求一种简单且快速提取和纯化许旺细胞的方法。方法:实验组大鼠在无菌条件下切断坐骨神经远端,使坐骨神经在体内预变性;对照组大鼠的坐骨神经不予任何处理。术后7d切取两组坐骨神经,采用混合酶消化、组织块移植法提取许旺细胞;通过低酶消化、2次接种差速贴壁法分离纯化许旺细胞。相差显微镜下观察细胞形态,并行免疫荧光染色鉴定;计算细胞纯度;MTT法检测细胞增殖能力。结果与结论:实验组培养第7天可见典型呈双极或三极的许旺细胞,细胞间建立连接;对照组细胞突起较短,与周围细胞较少关联。S-100免疫荧光染色后,细胞呈阳性绿色表达。实验组细胞增殖较快,第15天迅速形成漩涡状,成纤维细胞数量相对较少,细胞纯度达96.1%;对照组细胞在培养过程中增殖较缓慢,成纤维细胞数量多,细胞纯度较低。MTT法检测结果显示,原代培养时两组许旺细胞增殖能力均较弱;传代后与对照组比较,实验组许旺细胞增殖能力明显增强(P<0.05或0.01),第三四代达峰值。结果证实体内预变性、体外混合酶消化、组织块移植结合低酶消化、双差速贴壁分离许旺细胞是一种简便、快速的提取纯化许旺细胞的方法。
BACKGROUND: Schwann cell transplantation can change the microenvironment of local injury, is conducive to the repair of neural trauma, to obtain a large number of high-purity, proliferation-competent Schwann cells is the key to study. Objective: To explore a simple and rapid extraction and purification of Schwann cells. Methods: The rats in experiment group were cut off the distal end of sciatic nerve under aseptic conditions, the sciatic nerve was pre-denatured in vivo, and the sciatic nerve in control group was not treated. Two groups of sciatic nerve were cut out at 7 days after operation, and mixed Schwann cells were digested by mixed enzyme and tissue-block transplantation. Schwann cells were isolated and purified by differential inoculation with low enzyme digestion. Cell morphology was observed under a phase contrast microscope and immunofluorescence staining was performed. Cell purity was calculated. MTT assay was used to detect cell proliferation. RESULTS AND CONCLUSION: Schwann cells with typical bipolar or tripolar morphology were found on the 7th day in the experimental group. The connections among the cells were established. In the control group, the protuberances of the cells were shorter and were less correlated with the surrounding cells. S-100 immunofluorescence staining, the cells were positive green expression. In the experimental group, the cells proliferated rapidly and became swollen rapidly on the 15th day. The number of fibroblasts was relatively small with the purity of 96.1%. The cells in the control group proliferated more slowly, the number of fibroblasts was more and the cell purity was lower . The results of MTT assay showed that the proliferation ability of Schwann cells in both groups were weaker in primary culture. Compared with the control group, the proliferation ability of Schwann cells in experimental group was significantly increased (P <0.05 or 0.01) Peak. The results show that in vivo pre-denaturation, in vitro mixed enzyme digestion, tissue block transplantation combined with low enzyme digestion, double differential adherent separation of Schwann cells is a simple and rapid method of extraction and purification of Schwann cells.