α晶体蛋白与视网膜神经节细胞膜结合的靶蛋白的探索研究
发布时间：2019-04-15 12:51
【摘要】：目的:通过研究外源性α晶状体蛋白与视网膜神经节细胞的结合部位并探索与其作用的靶蛋白,初步探讨α晶状体蛋白促进RGCs存活及轴突再生的分子机制,为视神经损伤后的临床治疗和研究提供理论依据。 方法: 1、外源性α晶体蛋白与大鼠视网膜神经节细胞结合的定位研究:通过蛋白的生物素化技术对外源性的α晶状体蛋白进行生物素标记后与RGCs孵育,再用免疫组织化学技术研究外源性α晶状体蛋白与RGCs的结合部位; 2、外源性α晶体蛋白与视网膜神经节细胞膜受体integrinα6作用关系的研究:通过受体结合位点封闭的原理、免疫组织化学技术、蛋白质相互作用Pull-Down技术及免疫印迹实验,间接和直接验证外源性α晶状体蛋白是否和RGC-5膜上受体integrinα6有作用关系; 3、应用SDS-GAGE电泳及质谱的方法对用Pull-Down技术捕获的蛋白进行分析。 结果: 1.外源性α晶状体蛋白结合RGCs的胞膜上; 2、α晶状体蛋白与视网膜神经节细胞膜受体integrinα6无作用关系。 3、SDS-GAGE电泳凝胶上有一个50KD左右的条带,将其质谱分析,结果中谷胱甘肽S-转移酶的评分大于系统显著性评价的标准。 结论: 1.外源性α晶状体蛋白是通过结合于RGCs的胞膜上发挥促进其存活及轴突生长作用的; 2.尽管层粘连蛋白-1通过作用于RGCs上integrinα6有明显的促进轴突生长的作用,且α晶状体蛋白可以通过作用于晶体上皮细胞上的integrinα6起抗凋亡作用,但通过本实验证实,在RGCs上,α晶状体蛋白与integrinα6无作用关系。 3.谷胱甘肽S-转移酶可能是外源性α晶状体蛋白的靶蛋白,但还需进一步的验证。
[Abstract]:Objective: to explore the molecular mechanism of 伪-crystallin in promoting the survival and axon regeneration of RGCs by studying the binding site of exogenous 伪-crystallin with retinal ganglion cells and exploring the target protein. It provides a theoretical basis for the clinical treatment and research of optic nerve injury. Methods: 1. Localization of the binding of exogenous 伪-crystallin to rat retinal ganglion cells: the exogenous 伪-crystallin was biotinylated and incubated with RGCs. The binding site of exogenous 伪-crystallin to RGCs was studied by immunohistochemistry. 2. Studies on the relationship between exogenous 伪-crystallin and receptor integrin 伪 6 in retinal ganglion cell membrane: through the principle of blocking receptor binding sites, immunohistochemical technique, protein-protein interaction Pull-Down technique and immunoblotting test, the relationship between exogenous 伪-crystallin and retinal ganglion cell membrane receptor Pull-Down 伪 6 was studied. Indirect and direct verification of the relationship between exogenous 伪-crystallin and RGC-5 membrane receptor integrin 伪 6; 3. The protein captured by Pull-Down was analyzed by SDS-GAGE electrophoresis and mass spectrometry. Results: 1. Exogenous 伪-crystallin binds to the membrane of RGCs. 2. There is no relationship between 伪-crystallin and retinal ganglion cell membrane receptor integrin 伪 6. (3) there was a band of 50KD on SDS-Gage electrophoresis gel. The results of MS analysis showed that the score of glutathione S-transferase was higher than the criterion of systematic significance evaluation. Conclusions: 1. Exogenous 伪-crystallin promotes its survival and axon growth by binding to the membrane of RGCs. Although laminin-1 can significantly promote axon growth by acting on integrin 伪 6 on RGCs, and 伪-crystallin can inhibit apoptosis by acting on integrin 伪 6 on lens epithelial cells, it is confirmed by this experiment that 伪-crystallin can inhibit apoptosis in RGCs. There was no relationship between 伪-crystallin and integrin 伪 6. 3. Glutathione S-transferase may be a target protein of exogenous 伪-crystallin, but it needs further verification.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.6
本文编号：2458169
[Abstract]:Objective: to explore the molecular mechanism of 伪-crystallin in promoting the survival and axon regeneration of RGCs by studying the binding site of exogenous 伪-crystallin with retinal ganglion cells and exploring the target protein. It provides a theoretical basis for the clinical treatment and research of optic nerve injury. Methods: 1. Localization of the binding of exogenous 伪-crystallin to rat retinal ganglion cells: the exogenous 伪-crystallin was biotinylated and incubated with RGCs. The binding site of exogenous 伪-crystallin to RGCs was studied by immunohistochemistry. 2. Studies on the relationship between exogenous 伪-crystallin and receptor integrin 伪 6 in retinal ganglion cell membrane: through the principle of blocking receptor binding sites, immunohistochemical technique, protein-protein interaction Pull-Down technique and immunoblotting test, the relationship between exogenous 伪-crystallin and retinal ganglion cell membrane receptor Pull-Down 伪 6 was studied. Indirect and direct verification of the relationship between exogenous 伪-crystallin and RGC-5 membrane receptor integrin 伪 6; 3. The protein captured by Pull-Down was analyzed by SDS-GAGE electrophoresis and mass spectrometry. Results: 1. Exogenous 伪-crystallin binds to the membrane of RGCs. 2. There is no relationship between 伪-crystallin and retinal ganglion cell membrane receptor integrin 伪 6. (3) there was a band of 50KD on SDS-Gage electrophoresis gel. The results of MS analysis showed that the score of glutathione S-transferase was higher than the criterion of systematic significance evaluation. Conclusions: 1. Exogenous 伪-crystallin promotes its survival and axon growth by binding to the membrane of RGCs. Although laminin-1 can significantly promote axon growth by acting on integrin 伪 6 on RGCs, and 伪-crystallin can inhibit apoptosis by acting on integrin 伪 6 on lens epithelial cells, it is confirmed by this experiment that 伪-crystallin can inhibit apoptosis in RGCs. There was no relationship between 伪-crystallin and integrin 伪 6. 3. Glutathione S-transferase may be a target protein of exogenous 伪-crystallin, but it needs further verification.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.6
【参考文献】
相关期刊论文 前3条
1 王晓芹;王一;;α晶体蛋白对视网膜神经节细胞作用的初步研究[J];第三军医大学学报;2006年19期
2 应希;王一;曾玉晓;吴燕;;大鼠视网膜神经节细胞中内源性α晶状体蛋白的表达及其变化规律[J];眼科新进展;2010年09期
3 李琪;严宏;;α-晶状体蛋白在眼科疾病中的作用研究进展[J];眼科研究;2010年11期
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