雪旺细胞体外分离培养及甲基强地松龙对雪旺细胞分泌功能影响的研究

发布时间：2018-06-03 17:01

本文选题：雪旺细胞 + 神经生长因子　；参考：《大连医科大学》2008年硕士论文

【摘要】： 神经再生室技术的出现,解决了短距离神经再生的问题。但是除自体神经移植外,其他人工修复技术尚无法解决长距离神经缺损的问题。尽管组织工程技术在不断发展,但体外构建的“人工神经”还没有从本质上超越再生室技术。其原因是组织工程中作为种子细胞的细胞老化、分泌能力下降等诸多问题。雪旺细胞是周围神经最主要的胶质细胞,也是最重要的种子细胞。雪旺细胞分泌多种神经营养因子在神经再生中发挥关键作用,但其分泌能力受诸多因素影响,甲基强地松龙(MP)被认为是一种通过抑制脂质过氧化而对神经细胞及其突起发挥保护作用的药物。目的: 1.采用新生SD大鼠的雪旺细胞作为周围神经组织工程的种子细胞,在体外分离培养,探索体外培养及增殖雪旺细胞的方法。 2.选取生长状况和纯度较好的第二代细胞,将经胰酶消化下来的细胞接种到六孔板中,以不同浓度的MP作用于雪旺细胞,以研究MP对雪旺细胞分泌功能影响。方法: 1.取SD大鼠的新生鼠双侧的坐骨、臂丛神经,剪碎至1mm3大小的组织块,双酶消化法进行分离培养,阿糖胞苷抑制成纤维细胞生长,低浓度胰酶快速消化传代进一步纯化雪旺细胞,用光镜进行形态学观察、S-100蛋白进行鉴定。 2.取生长状况较好的第二代细胞,将经胰酶消化下来的细胞接种到六孔板中,以不同浓度的MP(10~(-3)、10~(-4)、10~(-6)、10~(-8) mol/L)作用于雪旺细胞,作用24、48和72h,设立不加药物的空白对照组,并通过RT-PCR的方法半定量检测其分泌的NGF,以研究MP对雪旺细胞分泌功能的影响。结果: 1.通过上述方法分离培养获得了经S-100蛋白鉴定纯度为88%的第二代雪旺细胞,细胞数量为3.128×107/ml,细胞形态多数为梭形,细胞排列成典型的漩涡状或栅栏状。 2. RT-PCR方法检测后,经Lab works 4.60软件进行分析处理,以内参基因β-actin为基准分析各条带的相对密度值,应用Spass 10.0软件进行统计分析得出结论:10-8 mol/L的MP作用72h分泌的NGF与空白对照组比较及与其它浓度、时间组比较表达增加(p0.05); 10-3 mol/L的MP在各时间段分泌的NGF与空白对照组及其它浓度、时间组比较表达减少(p0.05)。结论: 1.新生SD大鼠坐骨、臂丛神经中分离培养雪旺细胞来源确实,取材方便,可以作为周围神经组织工程试验研究的种子细胞来源,采用双酶消化法、阿糖胞苷抑制成纤维细胞生长,可以获得较高纯度的雪旺细胞。 2.不同浓度的MP对雪旺细胞的分泌能力有不同的影响:高浓度(10~(-3) mol/L)的MP抑制雪旺细胞分泌NGF;长时间、低浓度(72 h,10~(-8) mol/L)的MP则促进雪旺细胞分泌NGF,故低浓度的MP可以提高雪旺细胞分泌NGF的能力,更好发挥雪旺细胞作为种子细胞的能力。
[Abstract]:The technique of nerve regeneration chamber solves the problem of short-distance nerve regeneration. But with the exception of autologous nerve transplantation, other artificial repair techniques can not solve the problem of long-distance nerve defect. Despite the development of tissue engineering technology, the in vitro construction of artificial nerve has not surpassed the regenerative chamber technology in essence. The reasons are the aging of cells as seed cells and the decline of secretion ability in tissue engineering. Schwann cells are the most important glial cells and seed cells of peripheral nerve. Schwann cells secrete a variety of neurotrophic factors that play a key role in nerve regeneration, but their secretion capacity is affected by many factors. MPM is considered to be a protective drug against nerve cells and its processes by inhibiting lipid peroxidation. Objective: 1. Schwann cells from newborn SD rats were isolated and cultured in vitro as seed cells for peripheral nerve tissue engineering to explore the methods of culture and proliferation of Schwann cells in vitro. 2. In order to study the effect of MP on the secretory function of Schwann cells, the second generation cells with good growth status and purity were inoculated into the Six-well plate with different concentrations of MP to study the effect of MP on the secretion of Schwann cells. Methods: 1. The sciatic bones and brachial plexus nerves of newborn SD rats were isolated and cultured by double enzyme digestion method, which were cut to the size of 1mm3. Cytarabine inhibited the growth of fibroblasts, and Schwann cells were further purified by rapid digestion and subculture of low concentration trypsin. The S-100 protein was identified by light microscope. 2. The cells digested by trypsin were inoculated into the six-well plate and treated with different concentrations of MPO10OOOON-3OOOTRONIDAE (10OOOTHYL) and Schewan cells (2448 and 72 h, respectively), and the control group was set up as a blank control group, and the control group was treated with different concentrations of MPO10OOOOOTHYL, and the control group did not add drugs to the cells for 2448 and 72 hours, and the cells were treated with different concentrations. The effect of MP on secretory function of Schwann cells was studied by semi-quantitative detection of NGF secreted by RT-PCR. Results: 1. The second generation of Schwann cells with the purity of 88% identified by S-100 protein was obtained by the method mentioned above. The number of cells was 3.128 脳 107 / ml, and most of the cells were fusiform, and the cells were arranged in a typical whirlpool or palisade shape. 2. After detection by RT-PCR method, the relative density of each band was analyzed by Lab works 4.60 software, using 尾 -actin as reference. Using Spass 10.0 software, it was concluded that the NGF secreted by 10 ~ (-8) mol/L at 72 h was higher than that in the control group and compared with other concentrations, and the expression of NGF in the time group was higher than that in the control group, and the NGF secreted by 10 ~ (-3) mol/L was higher than that in the blank control group and other concentrations. In the time group, the expression of P0. 05 was decreased. Conclusion: 1. Schwann cells isolated and cultured from the sciatic and brachial plexus of newborn SD rats can be used as seed cells for peripheral nerve tissue engineering research. Cytarabine can inhibit the growth of fibroblasts by double enzyme digestion. High purity Schwann cells can be obtained. 2. Different concentrations of MP have different effects on the secretory ability of Schwann cells: high concentration of MP can inhibit the secretion of NGF in Schwann cells for a long time. The low concentration of MP increased the secretion of NGF by Schwann cells, and the ability of Schwann cells as seed cells was improved by low concentration of MP.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329.2

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