许旺细胞提取与纯化的新方法

doi:10.3969/j.issn.1673-8225.2010.19.037

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3593-3596.doi: 10.3969/j.issn.1673-8225.2010.19.037

• 干细胞基础实验 basic experiments of stem cells • 上一篇下一篇

许旺细胞提取与纯化的新方法

张际绯¹，赵富生¹，武庚²，刘志新¹，李月珍¹

牡丹江医学院，¹组织学与胚胎学教研室，²临床技能中心，黑龙江省牡丹江市 157011

出版日期:2010-05-07 发布日期:2010-05-07
通讯作者: 李月珍，硕士，教授，牡丹江医学院组织学与胚胎学教研室，黑龙江省牡丹江市 157011
作者简介:张际绯，女，1957年生，黑龙江省牡丹江市人，汉族，2005年哈尔滨医科大学毕业，博士，教授，主要从事干细胞移植与组织修复机制方面的研究。
基金资助:
黑龙江省自然科学基金资助项目(D200559)

A new method of extracting and purifying Schwann cells

Zhang Ji-fei¹, Zhao Fu-sheng¹, Wu Geng², Liu Zhi-xin¹, Li Yue-zhen¹

1Department of Histology and Embryology, 2Department of Clinical Technology, Mudanjiang Medical College, Mudanjiang 157011, Heilongjiang Province, China

Online:2010-05-07 Published:2010-05-07
Contact: Li Yue-zhen, Master, Professor, Department of Histology and Embryology, Mudanjiang Medical College, Mudanjiang 157011, Heilongjiang Province, China liyuezhennv@163.com
About author:Zhang Ji-fei, Doctor, Professor, Department of Histology and Embryology, Mudanjiang Medical College, Mudanjiang 157011, Heilongjiang Province, China zhangjifei777@163.com
Supported by:
the Natural Science Foundation of Heilongjiang Province, No. D200559*

摘要/Abstract

摘要：

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7 d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。

关键词: 细胞培养, 提取, 纯化, 坐骨神经, 预变性, 许旺细胞

Abstract:

BACKGROUND:Schwann cells transplantation can change the local micro-environment and help to repair the injured neural tissue, so getting a large number of highly purified and active Schwann cells is the key of the study.
OBJECTIVE: To search for a simple and rapid method to extract and purify the Schwann cells.
METHODS: Rats were divided randomly into two groups, namely, in vivo pre-degeneration of sciatic nerve resection group and untreated control group, with 20 rats in each group. Under sterile conditions, the rat sciatic nerves were cut off at post-operative 7 days, Schwann cells were extracted by using mixed enzyme digestion and tissue mass transplantation; through low enzyme digestion and twice inoculation to differential adhesion, Schwann cells were purified. Cell morphology was observed under phase contrast microscope and identified by immunofluorescence staining; cell purity was calculated; MTT method assay was used to determine the capacity of cell proliferation.
RESULTS AND CONCLUSION: At 7 days of the culture, the experimental group showed the typical bipolar or tripolar Schwann cells, with connections between cells; in control group, cell processes were shorter and less associated with the surrounding cells. Following S-100 immunofluorescence staining, cells were positive for green expression. Cells proliferated rapidly in the experimental group and formed a swirling shape at 15 days, there were a relatively small number of fibroblasts, at the purity of 96.1%; in the control group, the cells proliferated slowly, with many fibroblasts at a low purity. MTT assay showed that primary cultured Schwann cell proliferated weakly in both groups; compared with the control group, the proliferation of subcultured Schwann cells in the experimental group was markedly increased (P < 0.05 or 0.01), and reached a peak 3-4 days later. The results confirmed that in vivo denaturing, in vitro hybrid enzyme digestion, tissue mass transplantation combined with low enzyme digestion, separation of double-differential adhesion of Schwann cells is a simple and rapid method to extract and purify Schwann cells.

中图分类号:

R394.2

张际绯，赵富生，武庚，刘志新，李月珍. 许旺细胞提取与纯化的新方法[J]. 中国组织工程研究, 2010, 14(19): 3593-3596.

Zhang Ji-fei, Zhao Fu-sheng, Wu Geng, Liu Zhi-xin, Li Yue-zhen. A new method of extracting and purifying Schwann cells[J]. Chinese Journal of Tissue Engineering Research, 2010, 14(19): 3593-3596.

参考文献

引言

材料方法

讨论

A minimum amount of nerve tissue is used to obtain highly purified Schwann cells within a short time. During the culture of Schwann cells, two points are crucial: ① Removing fibroblasts in nerve membrane. Peripheral nerve fibroblasts are mainly located in the epineurium and perineurium, so that it is important to remove membrane components, therefore, stripping the sticking-like epineurium as clean as possible under a dissecting microscope is the basis for successful removal of the fibroblasts. As for the repeated tissue mass transplantation, due to fibroblasts climbed faster than Schwann cells, a large number of tissue fibroblasts would be abandoned after each transferring tissue, the number of fibroblast cells is limited. In this experiment, tissue transplantation were adopted, fibroblast cells decreased significantly. Low enzyme double-differential adhesion, utilizing Schwann cells were more sensitive than fibroblasts for trypsin, 2.5 g/L trypsin /0.2 g/L EDTA were applied in this experiment for low concentration digestive enzymes, combined with twice 30-minute differential separation, fibroblasts contaminated were further removed, to achieve the purpose of purifying Schwann cells. In the present experiment, the process of fibroblast removal exhibits many advantages such as non-toxicity, active cell vitality and so on. There are many methods to remove fibroblasts at home and abroad, but they have advantages and disadvantages^[14-16], chemical reagent method is commonly used. In 1976, Wood et al^[17] first reported the use of anti-mitotic agent Ara-c for inhibiting fibroblast and achieving purification, Wei et al^[18] also used the Ara-c to inhibit fibroblasts. Brockes et al^[19] dissolved fiber cells by adding thymosin 1 antiserum in the culture medium, so as to purify Schwann cells. These methods greatly affect clinical application of the cells. ② Fixing tissue mass. In the process of repeatedly transplanting tissue, the ability to firmly stick to the bottom dish is the key of implant survival and culture success. Experimental protocol is to paste tissue on the culture dishes pre-coated with poly-lysine on the bottom, stayed for a moment, a little culture medium added and cultured for 4 hours, and then medium carefully added to soak tissue. The method is convenient, but requires great dexterity. After in vivo denaturing, Schwann cells vigorously proliferated. When peripheral nerves were resected, axonal and myelin completely collapsed due to Wallerian phenomenon, while Schwann cells proliferated and formed Bunger’s belt, guiding the growth of regenerated axons, Schwann cells have a very strong growth and reproductive capacity. So in the culture process, the activated Schwann cells were faster than normal Schwann cells regarding the adherent speed and proliferation speed. In addition, when peripheral nerve injuries, macrophage infiltration could be observed, due to the macrophages participate in production of Schwann cells mitosis factors, the proliferation of Schwann cells is also accelerated^[20]. In this process, Schwann cells secrete mitogenic factors and further enhance their own proliferation^[21], also inhibit the growth of fibroblasts^[22]. By means of comprehensive use of various measures, without any mitogenic factors or cell division inhibitors, adult Schwann cells of high purity and strong proliferative activity can be obtained within a short term, thus providing cell materials for tissue engineering repair of peripheral and central nervous system injury.

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许旺细胞提取与纯化的新方法

A new method of extracting and purifying Schwann cells

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