α-晶状体蛋白对脂多糖诱导的视神经星形胶质细胞增生、活化及分泌功能的抑制作用

Inhibitory effects of α-crystallin on optic nerve astrocytes proliferation, activation and secretion stimulated by lipopolysaccharide

背景视神经损伤后星形胶质细胞的活化和增生引起的局部胶质瘢痕形成是神经细胞轴突难以再生的主要原因之一。研究表明，α-晶状体蛋白能促进视网膜神经节细胞（RGCs）轴突的再生，且部分再生的轴突能够穿过胶质瘢痕区，故推测α-晶状体蛋白可能抑制胶质瘢痕的形成，从而对视神经发挥保护作用。目的探讨α-晶状体蛋白对视神经星形胶质细胞的活化及其分泌功能的影响。方法分离SPF级3-5日龄Long Evans大鼠的视神经组织，体外培养和纯化视神经星形胶质细胞，采用免疫荧光技术检测细胞中胶质纤维酸性蛋白（GFAP）的表达以鉴定培养的细胞。将培养的细胞分为3个组，正常对照组细胞用常规细胞培养液进行培养，脂多糖（LPS）刺激组在培养液中添加5μg／ml LPS，α-晶状体蛋白干预组在培养液中添加5μg／ml LPS和1×10^-4g／L α-晶状体蛋白，各组细胞均继续培养24h。采用细胞计数试剂盒8（CCK-8）法检测各组视神经星形胶质细胞的增生情况（A值）；采用细胞免疫荧光法和Western blot法测定各组细胞中GFAP蛋白的表达；采用ELISA法检测各组细胞培养上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）质量浓度的变化。结果培养3-4代的细胞大小均匀，GFAP阳性细胞达95％以上。正常对照组、LPS刺激组和α-晶状体蛋白干预组细胞的A值分别为1．335±0．070、1．643±0．069和1．390±0．004，LPS刺激组A值明显高于正常对照组和α-晶状体蛋白干预组，差异均有统计学意义（t=3．315、3．681，均P〈0．05）。免疫荧光检测结果显示，LPS刺激组星形胶质细胞中的GFAP荧光明显强于正常对照组和α-晶状体蛋白干预组，且细胞胞体较正常对照组和α-晶状体蛋白干预组增大。Western blot法检测结果显示，LPS刺激组GFAP蛋白的相对表达量为0．851±0．076，高于正常对照组的0．786±0．091和α-晶状体蛋白干预组的0．569±0．049，其中α-晶状体蛋白干预组细胞中GFAP蛋白的相对表达量较LPS刺激组明显下降，差异有统计学意义（t=3．115，P〈0．01）。LPS刺激组TNF-α和IL-1β质量浓度明显高于正常对照组和α-晶状体蛋白干预组，差异均有统计学意义（均P〈0．05）。结论α-晶状体蛋白能抑制LPS诱导的视神经星形胶质细胞的增生、活化和炎性因子的释放。

Background Glial scaring induced by the activation and proliferation of astrocytes after optical nerve damage is one of causes of neural axons difficult to regeneration. Researches showed that α-crystallin can promote the regeneration and pass through scaring zone of retinal ganglion cells （RGCs） axons, and we speculate α-crystallin protect optical nerve tissue against scaring process. Objective This study was to investigate the influence of α-crystallin for the activation and secretion of inflammatory factors of astrocytes. Methods Optical nerver tissue was isolated from 3-5 day-old SPF Long Evans rats to culture and purify astrocytes. The cells were identified by detecting the expression of glial fibrillary acidic protein （GFAP） with immunofluorescence technique. The cells were cultured with regular culture medium in the normal control group, and 5 μg/ml lipopolysaccharides （LPS） was added in the LPS group,while 5 μg/ml LPS and 1×10^-4 g/L α-crystallin were added in the α-crystallin group,and the cells were consecutively cultured for 24 hours. The proliferation （absorbance,A） of the ceils was assayed by cell counting kit-8 （CCK-8）. The expression of GFAP in the ceils was detected by immunofluorescence technique and quantitated by Western blot. The contents in the cell supernatants of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） were detected by ELISA. Results The morphology and size were well-proportioned in 3-4 generation of cells with the GFAP positive rate over 95%. The A values were 1. 335±0. 070,1. 643 ±0. 069 and 1. 390±0. 004 in the normal control group,LPS group and α-crystallin group,and the A values in the LPS group were significantly higher than those in the normal control group and α-crystallin group （t = 3. 315,3. 681, both at P〈0.05 ）. Immunofluorescence examination showed that the fluorescence intensity was evidently enhanced in the LPS group compared with the normal control group and α-crystallin group and presented the largest cell bodies in the LPS group. The relative expressions of GFAP in the cells were 0. 851±0. 076 in the LPS group, which were higher than those in the normal control group and α-crystallin group （0. 786±0. 091,0. 569±0. 049）. Compared between the LPS group and α-crystallin group, there is a significant difference between the two groups （t = 3. 115,P〈0.01）. In addition, compared with the LPS group, the contents of TNF-α and IL-1β in the suspensions were significantly reduced in the normal control group and α-crystallin group （ all at P〈0. 05）. Conclusions α-Crystallin protein can inhibit the activation and secretion of optic nerve astrocytes stimulated by LPS.