【摘要】：目的:通過研究外源性α晶狀體蛋白與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的結(jié)合部位并探索與其作用的靶蛋白,初步探討α晶狀體蛋白促進(jìn)RGCs存活及軸突再生的分子機(jī)制,為視神經(jīng)損傷后的臨床治療和研究提供理論依據(jù)。 方法: 1、外源性α晶體蛋白與大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞結(jié)合的定位研究:通過蛋白的生物素化技術(shù)對外源性的α晶狀體蛋白進(jìn)行生物素標(biāo)記后與RGCs孵育,再用免疫組織化學(xué)技術(shù)研究外源性α晶狀體蛋白與RGCs的結(jié)合部位; 2、外源性α晶體蛋白與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞膜受體integrinα6作用關(guān)系的研究:通過受體結(jié)合位點(diǎn)封閉的原理、免疫組織化學(xué)技術(shù)、蛋白質(zhì)相互作用Pull-Down技術(shù)及免疫印跡實(shí)驗(yàn),間接和直接驗(yàn)證外源性α晶狀體蛋白是否和RGC-5膜上受體integrinα6有作用關(guān)系; 3、應(yīng)用SDS-GAGE電泳及質(zhì)譜的方法對用Pull-Down技術(shù)捕獲的蛋白進(jìn)行分析。 結(jié)果: 1.外源性α晶狀體蛋白結(jié)合RGCs的胞膜上; 2、α晶狀體蛋白與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞膜受體integrinα6無作用關(guān)系。 3、SDS-GAGE電泳凝膠上有一個(gè)50KD左右的條帶,將其質(zhì)譜分析,結(jié)果中谷胱甘肽S-轉(zhuǎn)移酶的評分大于系統(tǒng)顯著性評價(jià)的標(biāo)準(zhǔn)。 結(jié)論: 1.外源性α晶狀體蛋白是通過結(jié)合于RGCs的胞膜上發(fā)揮促進(jìn)其存活及軸突生長作用的; 2.盡管層粘連蛋白-1通過作用于RGCs上integrinα6有明顯的促進(jìn)軸突生長的作用,且α晶狀體蛋白可以通過作用于晶體上皮細(xì)胞上的integrinα6起抗凋亡作用,但通過本實(shí)驗(yàn)證實(shí),在RGCs上,α晶狀體蛋白與integrinα6無作用關(guān)系。 3.谷胱甘肽S-轉(zhuǎn)移酶可能是外源性α晶狀體蛋白的靶蛋白,但還需進(jìn)一步的驗(yàn)證。
[Abstract]:Objective: to explore the molecular mechanism of 偽-crystallin in promoting the survival and axon regeneration of RGCs by studying the binding site of exogenous 偽-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 偽-crystallin to rat retinal ganglion cells: the exogenous 偽-crystallin was biotinylated and incubated with RGCs. The binding site of exogenous 偽-crystallin to RGCs was studied by immunohistochemistry. 2. Studies on the relationship between exogenous 偽-crystallin and receptor integrin 偽 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 偽-crystallin and retinal ganglion cell membrane receptor Pull-Down 偽 6 was studied. Indirect and direct verification of the relationship between exogenous 偽-crystallin and RGC-5 membrane receptor integrin 偽 6; 3. The protein captured by Pull-Down was analyzed by SDS-GAGE electrophoresis and mass spectrometry. Results: 1. Exogenous 偽-crystallin binds to the membrane of RGCs. 2. There is no relationship between 偽-crystallin and retinal ganglion cell membrane receptor integrin 偽 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 偽-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 偽 6 on RGCs, and 偽-crystallin can inhibit apoptosis by acting on integrin 偽 6 on lens epithelial cells, it is confirmed by this experiment that 偽-crystallin can inhibit apoptosis in RGCs. There was no relationship between 偽-crystallin and integrin 偽 6. 3. Glutathione S-transferase may be a target protein of exogenous 偽-crystallin, but it needs further verification.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R774.6

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