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目的:探讨雪旺细胞(Schwann cells,SC)源营养神经活性物质(SC derived neurotrophic activity,SCNA)对体外培养视网膜节细胞正常及缺气损伤环境中存活及生长相关蛋白(GAP)_(43)表达的作用。方法:培养乳鼠SC,制备不同蛋白浓度的SCNA,加入原代培养的视网膜细胞中,MTT法检测SCNA活性培养荧光金逆行标记的新生3d的SD乳鼠视网膜细胞,接种于24孔板中。培养第2d时SCNA作用组培养液中加入300mg/l。SCNA,对照组不加;培养第5d缺气损伤组在培养液表面加入液体石蜡造成缺气损伤。12h后去除液体石蜡,观察细胞形态和计数荧光金逆行标记的视网膜节细胞(retinal ganglion cell,RGC)。Western Blot法分析SCNA对视网膜细胞GAP_(43)表达的影响。结果:SCNA能促进培养视网膜细胞的存活,并有蛋白浓度依赖关系。加SCNA后,视网膜细胞生长明显旺盛,悬浮的死细胞较少,RGC数量明显多于视网膜细胞单纯培养组(F=62.89,P<0.01)。缺气损伤组视网膜细胞出现肿胀改变,加有SCNA缺气损伤组细胞形态大致正常,RGC数与对照组相比有显著性差异(F=49.27,P<0.01)。GAP_(43)的表达在视网膜细胞正常及缺气损伤SCNA作用组上调。结论:SCNA对培养RGC具有明显营养作用,并上调GAP_(43)的表达。在培养液中加入SCNA,可以减轻“缺气”造成的损伤,提高体外培?
OBJECTIVE: To investigate the effect of SC derived neurotrophic activity (SCNA) on the survival and growth-related protein (GAP) in cultured retinal ganglion cells (RGCs) The role of expression. Methods: The SCNA of neonatal rat was cultured and SCNA of different protein concentration was prepared and added into primary cultured retinal cells. The retinal cells of SD neonatal SD rat retrogradely labeled with fluorescent gold were detected by MTT assay and seeded into 24-well plates. On the second day, 300mg / l SCNA was added to the culture medium. SCNA, the control group did not add; fifth day training group lack of air injury liquid paraffin on the surface of the culture resulting in a lack of air injury. After 12h, liquid paraffin was removed and cell morphology and retrograde labeling of fluorescein retinal ganglion cells (RGC) were observed. The effect of SCNA on the expression of GAP_ (43) in retinal cells was analyzed by Western Blot. Results: SCNA can promote the survival of cultured retinal cells, and the protein concentration-dependent relationship. After SCNA, retinal cell growth was obvious, fewer dead cells were suspended, and the number of RGCs was more than that of retinal cell culture group (F = 62.89, P <0.01). The retinal cells in the lack of air group showed swollen changes. The morphology of RGCs in the SCNA group was similar to that in the control group (F = 49.27, P <0.01). The expression of GAP_ (43) was upregulated in the SCNA-treated group with retinal cell injury and lack of air. CONCLUSION: SCNA has a significant nutritional effect on cultured RGCs and up-regulates the expression of GAP_ (43). By adding SCNA in the culture medium, can reduce the “lack of gas” caused by injury and improve in vitro culture?