大鼠耳蜗雪旺细胞体外培养、纯化及增殖的实验研究

发布时间：2018-06-06 07:06

本文选题：耳蜗 + 雪旺细胞　；参考：《第四军医大学》2012年硕士论文

【摘要】：感音神经性聋是临床工作中最常见的疾病，其发病机理和治疗方法仍在不断的探索中，目前认为主要病变部位在初级传入螺旋神经元（SGN）和耳蜗毛细胞。哺乳动物螺旋神经元和耳蜗毛细胞损伤后几乎不能再生，那么如何修复受损的SGN，如何使SGN增强在复杂的病理状态下的修复能力，如何使SGN在损伤后的特殊微环境里提高其存活率是感音神经性聋的研究方向之一。雪旺细胞是从胚胎时期周围神经系统的神经嵴前体细胞演化而来的一种胶质细胞。周围神经受损后其远端轴突和髓鞘崩解,此时雪旺细胞可以大量增殖并且吞噬远端轴突和崩解的变性坏死的碎片。雪旺细胞还可以分泌神经生长因子NGF和神经营养因子NTF，因此雪旺细胞具有可以增加神经元的存活率、促进细胞轴突生长和提高神经元再生的能力。大量研究显示，移植雪旺细胞能够促进脊髓、周围神经损伤的修复并具有加快中枢神经系统脱髓鞘性疾病功能恢复的作用。然而，目前尚无关于耳蜗雪旺细胞和螺旋神经元轴突的相互作用机制的研究报道和关于耳蜗雪旺细胞与外周神经系统的其他雪旺细胞之间的相似性和差异性的报道。而弄清楚以上问题的关键在于纯化和定性耳蜗雪旺细胞。所以本研究致力于进行大鼠仔鼠耳蜗雪旺细胞的体外培养、纯化的试验，并配制一种能使耳蜗雪旺细胞快速增殖的培养液配方。实验一大鼠耳蜗雪旺细胞体外培养和纯化选用1-3d SD大鼠，无菌条件下暴露双侧听泡，在高倍镜下仔细剥离蜗壳，开放耳蜗，完整取出耳蜗组织，分离并且除去膜蜗管外侧壁的血管纹和基底膜组织，，然后剪碎。用0.25%的胰蛋白酶消化，用胎牛血清中止消化，离心以后加入DMEM/F12培养液培养。3-5天后对细胞应用免疫磁珠阳性分选方法进行纯化，培养2天后进行传代接种，培养过程中对提纯后的大鼠耳蜗雪旺细胞进行形态学观察、并绘制其生长曲线，采用细胞免疫荧光染色对细胞进行S-100免疫荧光鉴定并且计算细胞纯度。结果：分离培养后所得的细胞即为雪旺细胞；利用免疫磁珠阳性分选法对培养所得的细胞进行纯化，纯化后的大鼠耳蜗雪旺细胞纯度为97％±1.2％。结论：免疫磁珠法是一种有效的分离纯化新生大鼠仔鼠耳蜗雪旺细胞的方法。所得雪旺细胞活力强、纯度高，可以用于耳蜗雪旺细胞与螺旋神经节轴突的生长和再生等相关研究。实验二耳蜗雪旺细胞增殖的试验取生长良好的第三代经免疫磁珠分离纯化的耳蜗雪旺细胞，消化后制成细胞浓度为1×10~4/ml的单细胞悬液，接种在96孔板上，每块6×12孔，每孔加入200μl细胞悬液，共接种10个样本。将硫酸乙酰肝素蛋白多糖HSPG和碱性成纤维细胞生长因子bFGF配置成不同浓度的的培养液。37℃，5%CO_2培养，每日固定时间取一块板，连续取10天，用激发波长为490nm的酶联免疫测试仪测吸光度值，计算当天各组A值均数，以光吸收值为纵轴，时间为横轴，绘制六组耳蜗雪旺细胞的生长曲线。结果：B、C、D、E、F组优于空白对照组A，有统计学意义（P0.05）；B、C、D组之间无明显差别，无统计学意义；E、F组优于B、C、D组，有统计学意义；E、F组之间无明显差别，无统计学意义。这说明用含硫酸乙酰肝素蛋白多糖HSPG和碱性成纤维细胞生长因子bFGF的细胞培养液能使耳蜗雪旺细胞快速增殖,其中50ng/mlHSPG+50ng/mlbFGF生长最旺盛，A、B、C、D四组在第7天达到分裂顶峰，E、F组在第9天达到分裂顶峰。
[Abstract]:Sensorineural deafness is the most common disease in clinical work. Its pathogenesis and treatment methods are still in constant exploration. At present, the main lesions are found in primary afferent spiral neurons (SGN) and cochlear hair cells. It is almost impossible to regenerate mammalian spiral neurons and cochlear hair cells, and how to repair damaged SGN, How to enhance the ability to repair SGN in a complex pathological state and how to improve the survival rate of SGN in a special microenvironment after injury is one of the research directions for sensorineural hearing loss. Schwann cells are a glial cell evolved from the neural crest precursor cells from the peripheral nervous system in the embryonic period. With the disintegration of the distal axon and myelin sheath, Schwann cells can proliferate and phagocytic fragments of the distal axons and disintegrating degeneration and necrosis. Schwann cells can also secrete nerve growth factor NGF and neurotrophic factor NTF. Therefore, Schwann cells can increase the survival rate of neurons, promote cell axon growth and increase neurons. A large number of studies have shown that the transplantation of Schwann cells can promote the repair of spinal cord, peripheral nerve damage and accelerate the function recovery of the demyelinating disease of the central nervous system. However, there is no research report on the interaction mechanism of cochlear Schwann cells and spiral neuron axons and the cochlear Schwann cells. The similarity and difference between the other Schwann cells of the peripheral nervous system is reported. The key to the above problem is to purify and determine the cochlear Schwann cells. Therefore, this study is devoted to the culture and purification of the cochlear Schwann cells in rat cochlea, and to make a rapid proliferation of the cochlear Schwann cells. The formula of culture medium.
In vitro culture and purification of rat Schwann cells from cochlea
1-3D SD rats were exposed to bilateral auditory vesicles under sterile conditions. The cochlea was carefully stripped under high magnification, cochlear was opened and the cochlear tissue was removed completely. The vascular and basal membrane tissues of the outer wall of the cochlear tube were separated and removed, and then shredded. Digestion with 0.25% trypsin, fetal bovine serum digestion and DMEM/F12 culture after centrifugation were added. After culture.3-5 days, the immunomagnetic beads positive separation method was purified and cultured for 2 days. During the culture, the cultured rat cochlear Schwann cells were observed, and the growth curves were plotted. Cell immunofluorescence staining was used to identify the cells by S-100 immunofluorescence and to calculate the cell purity. Results: the cells obtained from the isolation and culture were Schwann cells, and the cultured cells were purified by immunomagnetic beads positive separation. The purity of the purified cochlear Schwann cells after purification was 97% + 1.2%. conclusion: the immunomagnetic bead method was an effective method to separate and purify the cochlear Schwann cells of newborn rat cochlea. The vitality and purity of the cells are strong and can be used for the study of the growth and regeneration of the Schwann cells and the spiral ganglion axons.
Experiment two the proliferation of Schwann cells in the cochlea
A well grown third generation of cochlear Schwann cells were isolated and purified by immunomagnetic beads. After digestion, a single cell suspension with a concentration of 1 x 10~4/ml was prepared, inoculated on 96 orifice plates, each 6 x 12 holes, and 200 mu L cell suspension was added to each hole, and 10 samples were inoculated. The heparan sulfate proteoglycan HSPG and basic fibroblast growth factor bFG were taken. F was formed into a medium of different concentration of.37, 5%CO_2 culture and a plate for 10 days at a fixed time. The absorbance values were measured by an enzyme linked immunosorbent assay with the excitation wavelength of 490nm. The average number of A values in each group was calculated on the same day. The growth curve of six groups of cochlear Schwann cells was drawn with light absorption value as a longitudinal axis and a horizontal axis. Results: B, C, D, E, F group was superior to blank control group A, with statistical significance (P0.05); there was no significant difference between group B, C and D, E, F group was superior to B, C, D group, with statistical significance; E, there was no significant difference between B, C, D group. This shows that the cell culture with the egg white polysaccharide sulfate containing heparin sulfate and basic fibroblast growth factor The liquid can rapidly proliferate the cochlear Schwann cells, in which the 50ng/mlHSPG+50ng/mlbFGF growth is the most vigorous. The four groups of A, B, C, D reach the peak of division at seventh days, E, and the F group reach the split peak at ninth days.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764.43

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