低濃度血清法培養(yǎng)純化新生大鼠雪旺細胞
發(fā)布時間：2019-03-21 11:08
【摘要】：雪旺細胞(Schwann cell,SCs)是周圍神經(jīng)系統(tǒng)一種重要的膠質(zhì)細胞,能夠形成有髓和無髓神經(jīng)纖維的神經(jīng)內(nèi)膜組織,具有多種的生理學功能,增生的雪旺細胞能夠產(chǎn)生和分泌多種的能夠促進神經(jīng)損傷后神經(jīng)再生修復的神經(jīng)營養(yǎng)因子和細胞粘附分子等。在神經(jīng)的再生過程中,雪旺細胞可以通過吞噬變性的軸突和髓鞘碎屑、分泌多種神經(jīng)營養(yǎng)因子(包括NGF,BDNF等)、分泌層粘連蛋白等多種基底膜成份等途徑,為近端軸突的再生提供一個良好的再生微環(huán)境,引導軸突再生。隨著組織工程技術的發(fā)展,在能夠構建神經(jīng)導管的支架材料上種植雪旺細胞,移植人體可以促進軸突的快速生長,進一步的促進神經(jīng)修復,但是如何能夠快速與大量的獲得純度高、狀態(tài)好的雪旺細胞是移植成功與否的關鍵。 目的:1.探討分離,培養(yǎng),SD大鼠雪旺細胞的方法。2.利用低濃度血清培養(yǎng)基純化體外培養(yǎng)的新生大鼠的雪旺細胞。3.觀察和檢測低濃度血清處理后的雪旺細胞生物學性狀和神經(jīng)因子分泌能力的影響。 方法:1.取新生3-7日的SD大鼠雙側(cè)坐骨神經(jīng),顯微鏡下將神經(jīng)剪碎至1mm3左右大小的神經(jīng)碎塊,利用雙酶消化法分離培養(yǎng),光鏡觀察細胞形態(tài)及細胞生長情況。6天后分別以含有胎牛血清濃度為10%、2%及無血清DMEM培養(yǎng)基處理原代培養(yǎng)的雪旺細胞6天后,終止培養(yǎng),并收集細胞。2.分別取原代培養(yǎng)及處理過后的雪旺細胞,采用免疫組化技術:以SABC方法標記雪旺細胞的特異性標記性蛋白S100,并在200倍顯微視野下以細胞計數(shù)方式分別統(tǒng)計雪旺細胞及成纖維細胞數(shù),從而得到雪旺細胞的純度即雪旺細胞數(shù)/雪旺細胞+成纖維細胞數(shù),獲得數(shù)據(jù)以t檢驗方法進行比較;流式細胞儀檢測細胞增值比率:在終止細胞培養(yǎng)時,以0.25%胰酶消化細胞,并以70%無水乙醇固定細胞,利用流式細胞儀分析得到S期、G2期的比率并進行統(tǒng)計學分析;利用RT-PCR法NGF、BDNF的mRNA在細胞內(nèi)的表達情況:在終止處理后,以Trizol提取液方法提取細胞的總RNA以β-肌動蛋白(β-actin)做內(nèi)標準,利用反轉(zhuǎn)錄-多聚酶鏈反應(RT-PCR)對A組和B組的NGF、BDNF的mRNA進行擴增,取PCR產(chǎn)物行l(wèi)%瓊脂糖凝膠電泳,測量其灰度值,分別計算NGF、BDNF和內(nèi)標基因(β-actin)PCR產(chǎn)物的灰度比值半定量和NGF、BDNF;統(tǒng)計學分析:數(shù)據(jù)采用SPSS11.0統(tǒng)計軟件進行統(tǒng)計分析。 結果:1.以2%濃度血清DMEM培養(yǎng)基分離培養(yǎng)純化后,雪旺細胞的狀態(tài)好,統(tǒng)計學結果分析表明其純度達到96.9%以上。2.處理后的細胞S100蛋白表達穩(wěn)定,各組之間的細胞周期S期、G2期的比率相同、NGF及BDNF分泌能力未受到影響。 結論:利用低濃度血清方法處理原代培養(yǎng)的雪旺細胞,處理后雪旺細胞的生物學性狀未受到影響。此方法簡化了雪旺細胞的培養(yǎng)純化方法,有效地控制了成纖維細胞污染。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
本文編號：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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329
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