应用免疫磁珠法分离提纯雪旺细胞及其增殖的实验研究

发布时间：2017-12-31 12:26

本文关键词：应用免疫磁珠法分离提纯雪旺细胞及其增殖的实验研究　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 雪旺细胞(Schwann cells,SCs)是周围神经系统的主要胶质细胞,在周围神经损伤修复中起决定性作用,它能分泌多种神经营养因子促进神经轴突的发芽再生;为再生轴突的延伸和生长提供隧道。所以,目前认为雪旺细胞在周围神经损伤修复及神经组织工程中有良好的应用前景。在体外培养中,成纤维细胞的增殖速度远远快于雪旺细胞,使雪旺细胞的分离和提纯因成纤维细胞的污染而变得困难。因此如何快速获取大量、高纯度、高活性雪旺细胞目前仍是制约雪旺细胞移植的关键。本实验的目的是通过免疫磁珠法分离及提纯雪旺细胞,并配置一种能够使雪旺细胞快速增殖的培养液配方。实验一应用免疫磁珠法分离及提纯雪旺细胞选用4～7d SD大鼠,无菌条件下取双侧坐骨神经,长约1cm,解剖镜下显微剥离去除神经外膜,获得神经束。将神经束剪碎至约1mm3大小颗粒。采用双酶二次消化法消化,加入胎牛血清中止消化后,离心并加入DF12培养液培养。7d后应用免疫磁珠法对细胞进行纯化培养,培养2d后进行传代接种。培养过程中对细胞进行形态学观察,计数及活力测定;MTT法绘制细胞生长曲线,采用免疫细胞化学荧光染色对细胞进行鉴定并且计算所得细胞纯度。结果:经S-100及免疫荧光鉴定所得细胞为雪旺细胞;采用免疫磁珠法能对培养所得雪旺细胞进行纯化。纯化后雪旺细胞活力为96%,纯度98%,培养2d后就可以传代。结论:免疫磁珠法可以用于雪旺细胞的纯化培养,所得雪旺细胞纯度高,活力强,能满足组织工程人工神经的需要。实验二雪旺细胞增殖的实验研究取生长良好的第二代雪旺细胞,经消化制成单细胞悬液,接种于96孔培养板,接种密度为1×104/ml,每块6×12孔,200ul/孔,共接种9个样本。实验分成6组(每组12孔):A组为空白对照组(基础培养液);B组为含50ng/ml碱性成纤维细胞生长因子(basic fibroblast growth factor bFGF)培养液组;C组为含50ng/ml硫酸乙酰肝素蛋白多糖(Heparin sulfate proteoglycan protein,HSPG)培养液组;D组为含100ng/ml HSPG细胞培养液组;E组为含50ng/mlbFGF +50ng/ml HSPG细胞培养液组;F组为含50ng/ml bFGF +100ng/mlHSPG细胞培养液组。37oC,5%CO2培养,每日固定时间取板1块(每间隔24h取板1块),酶联免疫检测仪测吸光度值(A值/OD值),激发波长:490nm,计算当日各组A值均数,以时间为横轴,光吸收值为纵轴绘制生长曲线。结果:B、C、D、E、F组均优于对照组,有统计学意义(P0.05);B、C、D组没有明显差别,无统计学意义(P0.05);E、F组均优于B、C、D组,有统计学意义(P0.05);E、F组没有明显差别,无统计学意义(P0.05)。结论:用含bFGF + HSPG细胞培养液能够使雪旺细胞快速增殖;含50ng/mlbFGF +100ng/ml HSPG细胞培养液(F组)生长最旺盛,E、F组第9天到分裂顶峰期;A、B、C、D第7天到分裂顶峰期。
[Abstract]:Schwann cells (Schwann cells SCs) is the main glial cells of the peripheral nervous system, play a decisive role in the repair of peripheral nerve injury, it can sprouting secretion of various neurotrophic factors to promote neurite extension; axonal regeneration and growth of tunnel. So, the Schwann cell has a good application prospect in the surrounding repair of nerve injury and nerve tissue engineering. In vitro, fibroblast proliferation is much faster than that of Schwann cells, separation and purification of Schwann cells and fibroblasts become difficult because of pollution. So how to obtain a large amount, high purity, high activity of Schwann cells is the key restriction of Schwann cell transplantation. The purpose of this experiment is through immunomagnetic separation and purification of Schwann cells, and the allocation of a party can make the medium with the rapid proliferation of Schwann cells.
Isolation and purification of Schwann cells by immunomagnetic beads
With 4 ~ 7d SD rats were dissected under sterile conditions and bilateral sciatic nerve, the length of about 1cm, under the anatomical microscope microscopic stripping removal of epineurium, obtain the nerve bundle. The nerve bundle was cut to about the size of 1mm3 particles. The digestion of two double enzyme digestion method, adding fetal bovine serum suspension after digestion with DF12 culture application of immunomagnetic liquid culture after.7d cells were purified and cultured on centrifugation and cultured 2D were inoculated culture. To observe the morphology of cells during the process of counting and determination of activity; cell growth curve was drawn by MTT staining, the cells were identified and calculated the purity of cells by immunocytochemistry. Results: after fluorescence S-100 and immunofluorescence to identify the cells into Schwann cells; using immunomagnetic beads can be used to purify the Schwann cells. The purified Schwann cell activity was 96%, the purity of 98%, after 2D culture can be passaged. Conclusion: The immunomagnetic bead method can be used in the purification and culture of Schwann cells. The purity and vitality of Schwann cells can meet the needs of the tissue engineering artificial nerve.
Experimental study on the proliferation of Schwann cells in experiment two
The second generation of good growth of Schwann cells, digested into single cell suspension, were seeded in 96 well plates, inoculum density is 1 * 104/ml, each 6 x 12 holes, 200ul/ hole, together with 9 samples. The experiment was divided into 6 groups (each 12 hole): group A (Foundation medium); group B growth factor 50ng/ml containing basic fibroblast (basic fibroblast growth factor bFGF) medium group; C group containing 50ng/ml heparan sulfate proteoglycan (Heparin Sulfate Proteoglycan Protein, HSPG) medium group; group D nutrient solution containing 100ng/ml group HSPG E group cultured cell culture; liquid group containing 50ng/mlbFGF +50ng/ml HSPG cells; group F medium group.37oC, containing 50ng/ml bFGF +100ng/mlHSPG 5%CO2 cells were cultured, fixed time daily from 1 boards (every 24h 1 boards), the absorbance measured the enzyme immunoassay (A / OD), excitation wavelength: 490nm, calculate the date the A values of each group The average number of time as the horizontal axis, the light absorption value of growth curve for the vertical axis. Results: B, C, D, E, F group was better than the control group, there was statistical significance (P0.05); B, C, D group have no obvious difference, no statistical significance (P0.05); E, F were significantly higher than those of B, C, D group, with statistical significance (P0.05); E F group, no significant difference was not statistically significant (P0.05).
Conclusion: using bFGF + HSPG cell culture medium can make Schwann cells proliferate rapidly. The growth of 50ng/mlbFGF +100ng/ml HSPG cell culture medium (F group) is the most vigorous, E, F group ninth days to the peak stage of division, A, B, C, and seventh days to the peak of division.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R651;R329

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