低浓度血清法培养纯化新生大鼠雪旺细胞
发布时间：2019-03-21 11:08
【摘要】：雪旺细胞(Schwann cell,SCs)是周围神经系统一种重要的胶质细胞,能够形成有髓和无髓神经纤维的神经内膜组织,具有多种的生理学功能,增生的雪旺细胞能够产生和分泌多种的能够促进神经损伤后神经再生修复的神经营养因子和细胞粘附分子等。在神经的再生过程中,雪旺细胞可以通过吞噬变性的轴突和髓鞘碎屑、分泌多种神经营养因子(包括NGF,BDNF等)、分泌层粘连蛋白等多种基底膜成份等途径,为近端轴突的再生提供一个良好的再生微环境,引导轴突再生。随着组织工程技术的发展,在能够构建神经导管的支架材料上种植雪旺细胞,移植人体可以促进轴突的快速生长,进一步的促进神经修复,但是如何能够快速与大量的获得纯度高、状态好的雪旺细胞是移植成功与否的关键。 目的:1.探讨分离,培养,SD大鼠雪旺细胞的方法。2.利用低浓度血清培养基纯化体外培养的新生大鼠的雪旺细胞。3.观察和检测低浓度血清处理后的雪旺细胞生物学性状和神经因子分泌能力的影响。 方法:1.取新生3-7日的SD大鼠双侧坐骨神经,显微镜下将神经剪碎至1mm3左右大小的神经碎块,利用双酶消化法分离培养,光镜观察细胞形态及细胞生长情况。6天后分别以含有胎牛血清浓度为10%、2%及无血清DMEM培养基处理原代培养的雪旺细胞6天后,终止培养,并收集细胞。2.分别取原代培养及处理过后的雪旺细胞,采用免疫组化技术:以SABC方法标记雪旺细胞的特异性标记性蛋白S100,并在200倍显微视野下以细胞计数方式分别统计雪旺细胞及成纤维细胞数,从而得到雪旺细胞的纯度即雪旺细胞数/雪旺细胞+成纤维细胞数,获得数据以t检验方法进行比较;流式细胞仪检测细胞增值比率:在终止细胞培养时,以0.25%胰酶消化细胞,并以70%无水乙醇固定细胞,利用流式细胞仪分析得到S期、G2期的比率并进行统计学分析;利用RT-PCR法NGF、BDNF的mRNA在细胞内的表达情况:在终止处理后,以Trizol提取液方法提取细胞的总RNA以β-肌动蛋白(β-actin)做内标准,利用反转录-多聚酶链反应(RT-PCR)对A组和B组的NGF、BDNF的mRNA进行扩增,取PCR产物行l%琼脂糖凝胶电泳,测量其灰度值,分别计算NGF、BDNF和内标基因(β-actin)PCR产物的灰度比值半定量和NGF、BDNF;统计学分析:数据采用SPSS11.0统计软件进行统计分析。 结果:1.以2%浓度血清DMEM培养基分离培养纯化后,雪旺细胞的状态好,统计学结果分析表明其纯度达到96.9%以上。2.处理后的细胞S100蛋白表达稳定,各组之间的细胞周期S期、G2期的比率相同、NGF及BDNF分泌能力未受到影响。 结论:利用低浓度血清方法处理原代培养的雪旺细胞,处理后雪旺细胞的生物学性状未受到影响。此方法简化了雪旺细胞的培养纯化方法,有效地控制了成纤维细胞污染。
[Abstract]:Schwann cell (Schwann cell,SCs) is an important glial cell in peripheral nervous system. It can form nerve intimal tissue with myelinated and unmyelinated nerve fibers and has many physiological functions. Proliferative Schwann cells can produce and secrete many kinds of neurotrophic factors and cell adhesion molecules which can promote nerve regeneration and repair after nerve injury. In the process of nerve regeneration, Schwann cells can secrete many kinds of neurotrophic factors (including NGF,BDNF, etc.), laminin and other basement membrane components through phagocytosis of degenerated axons and myelin debris. To provide a good microenvironment for regeneration of proximal axons and guide axon regeneration. With the development of tissue engineering technology, the implantation of Schwann cells on scaffolds capable of constructing nerve ducts can promote the rapid growth of axons and further promote nerve repair. But how to obtain high purity and good state Schwann cells quickly and in large quantities is the key to the success of transplantation. Purpose: 1. Methods for isolation and culture of Schwann cells from SD rats. Purification of neonatal rat Schwann cells cultured in vitro using low concentration serum medium. 3. The effects of low concentration serum on the biological characteristics of Schwann cells and neurofactor secretion were observed and detected. Methods: 1. The sciatic nerve of newborn 3-7 days SD rats was taken and the nerve fragments about the size of 1mm3 were cut under microscope and cultured by double enzyme digestion method. The morphology and cell growth of Schwann cells were observed under light microscope. After 6 days, Schwann cells were treated with fetal bovine serum (FBS) 10%, 2% and serum-free DMEM for 6 days, then the cultured cells were terminated and the cells were collected. Schwann cells were cultured and treated respectively. Immunohistochemical technique was used to label the specific marker protein S100 of Schwann cells by SABC method. The number of Schwann cells and fibroblasts were counted in 200-fold microscopic field of view, and the purity of Schwann cells was obtained, that is, Schwann cell number / Schwann cell fibroblast number. The data were compared with t-test method. Flow cytometry was used to detect the cell proliferation ratio: the cells were digested with 0.25% trypsin and immobilized with 70% ethanol at the end of cell culture. The ratio of S phase and G2 phase was obtained by flow cytometry and analyzed statistically. The expression of NGF,BDNF mRNA in cells by RT-PCR method: after the treatment was terminated, the total RNA was extracted by Trizol extraction method and 尾-actin was used as internal standard, and 尾-actin (尾-actin) was used as the internal standard to extract the total RNA from the cells. The mRNA of NGF,BDNF in group A and B was amplified by reverse transcription polymerase chain reaction (RT-PCR). The PCR products were analyzed by 1% agarose gel electrophoresis. The gray values of the products were measured and the NGF, values were calculated respectively. Semi-quantitative and NGF,BDNF; of BDNF and Internal Standard Gene (尾-actin) PCR Product Gray scale ratio) Statistical analysis: the data were analyzed by SPSS11.0 software. Results: 1. The Schwann cells were in good condition after isolation and purification with 2% serum DMEM medium. The results of statistical analysis showed that the purity of Schwann cells was more than 96.9%. The expression of S100 protein was stable after treatment. The ratio of cell cycle S-phase and G-2 phase was the same among the three groups. The secretion of NGF and BDNF were not affected. Conclusion: the primary cultured Schwann cells were treated with low concentration serum, and the biological characters of Schwann cells were not affected. This method simplifies the method of culture and purification of Schwann cells and effectively controls fibroblast contamination.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2444878
[Abstract]:Schwann cell (Schwann cell,SCs) is an important glial cell in peripheral nervous system. It can form nerve intimal tissue with myelinated and unmyelinated nerve fibers and has many physiological functions. Proliferative Schwann cells can produce and secrete many kinds of neurotrophic factors and cell adhesion molecules which can promote nerve regeneration and repair after nerve injury. In the process of nerve regeneration, Schwann cells can secrete many kinds of neurotrophic factors (including NGF,BDNF, etc.), laminin and other basement membrane components through phagocytosis of degenerated axons and myelin debris. To provide a good microenvironment for regeneration of proximal axons and guide axon regeneration. With the development of tissue engineering technology, the implantation of Schwann cells on scaffolds capable of constructing nerve ducts can promote the rapid growth of axons and further promote nerve repair. But how to obtain high purity and good state Schwann cells quickly and in large quantities is the key to the success of transplantation. Purpose: 1. Methods for isolation and culture of Schwann cells from SD rats. Purification of neonatal rat Schwann cells cultured in vitro using low concentration serum medium. 3. The effects of low concentration serum on the biological characteristics of Schwann cells and neurofactor secretion were observed and detected. Methods: 1. The sciatic nerve of newborn 3-7 days SD rats was taken and the nerve fragments about the size of 1mm3 were cut under microscope and cultured by double enzyme digestion method. The morphology and cell growth of Schwann cells were observed under light microscope. After 6 days, Schwann cells were treated with fetal bovine serum (FBS) 10%, 2% and serum-free DMEM for 6 days, then the cultured cells were terminated and the cells were collected. Schwann cells were cultured and treated respectively. Immunohistochemical technique was used to label the specific marker protein S100 of Schwann cells by SABC method. The number of Schwann cells and fibroblasts were counted in 200-fold microscopic field of view, and the purity of Schwann cells was obtained, that is, Schwann cell number / Schwann cell fibroblast number. The data were compared with t-test method. Flow cytometry was used to detect the cell proliferation ratio: the cells were digested with 0.25% trypsin and immobilized with 70% ethanol at the end of cell culture. The ratio of S phase and G2 phase was obtained by flow cytometry and analyzed statistically. The expression of NGF,BDNF mRNA in cells by RT-PCR method: after the treatment was terminated, the total RNA was extracted by Trizol extraction method and 尾-actin was used as internal standard, and 尾-actin (尾-actin) was used as the internal standard to extract the total RNA from the cells. The mRNA of NGF,BDNF in group A and B was amplified by reverse transcription polymerase chain reaction (RT-PCR). The PCR products were analyzed by 1% agarose gel electrophoresis. The gray values of the products were measured and the NGF, values were calculated respectively. Semi-quantitative and NGF,BDNF; of BDNF and Internal Standard Gene (尾-actin) PCR Product Gray scale ratio) Statistical analysis: the data were analyzed by SPSS11.0 software. Results: 1. The Schwann cells were in good condition after isolation and purification with 2% serum DMEM medium. The results of statistical analysis showed that the purity of Schwann cells was more than 96.9%. The expression of S100 protein was stable after treatment. The ratio of cell cycle S-phase and G-2 phase was the same among the three groups. The secretion of NGF and BDNF were not affected. Conclusion: the primary cultured Schwann cells were treated with low concentration serum, and the biological characters of Schwann cells were not affected. This method simplifies the method of culture and purification of Schwann cells and effectively controls fibroblast contamination.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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