刺参多糖对星形胶质细胞活化作用机制研究

On Sea Cucumber Polysaccharide in the Activation Mechanism of Astrocytes

目的探讨刺参多糖对星形胶质细胞的活化作用机制.方法选取新生Wistar大鼠24只,经胰酶消化分离可得星形胶质细胞;采用MTT法检测HS-4对细胞的毒副作用;采用伊红染色与免疫荧光染色检查细胞的形态学变化;采用Western blot法检测细胞特征蛋白GFAP和细胞周期调控蛋白Cyc DI的表达;采用BrdU法检测细胞的增殖;采用Transwell法检测细胞的迁移.结果 HS-4浓度在0.1～100μg/mL,作用时间为3,5 d时,细胞数量与对照组差异不显著,HS-4浓度在100μg/mL,作用时间为7 d时,细胞存活率明显低于对照组,差异显著;体外培养的细胞与HS-4单独作用,细胞形态无明显改变,HS-4与FGF-2联合作用后,细胞形态发生明显改变;星形胶质细胞的特征蛋白GFAP表达水平显著升高,HS-4在1μg/mL和5μg/mL时,GFAP表达升高了169%和183%;对照组细胞周期调控蛋白Cyc DI表达最低,FGF-2单独作用后,Cyc DI表达略有升高,当HS-4与FGF-2联合作用后,Cyc DI表达明显升高;对照组BrdU阳性率最低,占细胞总数的12.9%,FGF-2单独作用后,BrdU阳性率略有升高,占细胞总数的16.4%,与对照组差异不显著,HS-4浓度为1μg/mL和5μg/mL时与FGF-2联合作用,BrdU阳性率明显升高,占细胞总数的28.5%和56.1%,与对照组相比差异显著;静息状态星形胶质细胞无迁移,HS-4与FGF-2作用下星形胶质细胞迁移明显.结论 HS-4与FGF-2能有效诱导星形胶质细胞的活化,其活化机制主要是强化细胞增殖与迁移.

Objective To investigate the activation mechanism of sea cucumber polysaccharide on astrocytes. Method 24 Newborn Wistar rats were selected. Trypsin digestion were used to digest and separate tissues to obtain the astrocytes. The cytotoxie effect of HS-4 was detected by MTT. Morphological changes of cell were examined by eosin staining and immunofluorescence staining. The expression of the cell-specific protein GFAP and cell cycle regulatory protein Cyc DI were measured by Western blot method. The cell proliferation was observed by BrdU method. The migration of cells was shown by Transwell method. Results When HS-4 concentration was 0.1 -100 μg/mL,the reaction time was 3 d or 5 d,the difference in the number of cells was not significant compared with that of the control group. When HS-4 concentration was 100 μg/mL, the reaction time was 7 d, the cell survival rate was significantly lower than that of the control group, and the difference was significant. The change in the morphology of cells was not significant when HS-4 was used alone in vitro, but the change in the morphology of cells was significant after HS-d and FGF-2 were used at the same time. Astrocytes- specific GFAP expression increased significantly. When I-IS4 concentration was 1 μg/mL and 5 μg/mL, GFAP expression was increased by 169% and 183% ,respectively. The expression of cell cycle regulatory protein Cyc DI in the control group was lowest. Cyc DI expression increased slightly when FGF-2 was alone. When HS-4 and FGF-2 were used in combination, Cyc DI expression increased significantly. The positive rate of BrdU in the control group was the lowest, which accounted for 12.9% of total cells. The positive rate of BrdU increased slightly when FGF-2 was alone, which accounted for 16.4% of total cells. The difference was not obvious compared with that of the control group. When HS4 concentration was 1 ~g/mL and 5 μg/mL in combination with FGF-2 ,the positive rate of BrdU increased significantly, which accounted for 28.5% and 56.1% of total cells. The difference was significant compared with that of the control group. The resting astrocytes did not migrate, but the astrocyte treated with HS4 and FGF-2 migrated significantly. Conclusion HS-d and FGF-2 can effectively induce the activation of astrocytes,which can enhance the proliferation and migration of cells.