【摘要】：視神經(jīng)損傷是一種視功能損傷嚴(yán)重,預(yù)后極差的眼外傷類型和致盲原因。因?yàn)橹袠猩窠?jīng)系統(tǒng)組織損傷后不能再生,和缺乏修復(fù)功能,目前尚無(wú)確切的治療方式。我們的研究小組近十年的研究結(jié)果表明晶體損傷后釋放的晶體蛋白對(duì)視神經(jīng)損傷有修復(fù)和再生作用,這一現(xiàn)象為視神經(jīng)損傷的治療帶來(lái)了希望。近年的國(guó)內(nèi)外研究表明晶體蛋白中的αB-晶體蛋白是這一家族中最活躍的小分子蛋白,屬于熱休克蛋白家族,不僅可以對(duì)變性神經(jīng)組織有保護(hù)和促進(jìn)修復(fù)作用,還具有抑制炎癥、應(yīng)激反應(yīng),維護(hù)細(xì)胞骨架蛋白和骨架網(wǎng)絡(luò)穩(wěn)定性,及抵抗缺氧、高溫、反射線及藥物等因素誘導(dǎo)的細(xì)胞凋亡的作用。但由于αB-晶體蛋白不能從提取的天然α-晶體蛋白中分離出來(lái),重組人αB-晶狀體蛋白是開發(fā)應(yīng)用的唯一途徑。國(guó)外文獻(xiàn)報(bào)道重組αB-晶體蛋白用于治療多發(fā)性硬化動(dòng)物實(shí)驗(yàn)取得了良好的療效。而重組人αB-晶體蛋白全身應(yīng)用,是否具有視神經(jīng)保護(hù)作用未見文獻(xiàn)報(bào)道。由此,探討如何重組有高活性的人αB-晶體蛋白,明確重組人αB-晶體蛋白靜脈應(yīng)用,對(duì)視神經(jīng)保護(hù)作用,并對(duì)全身重要器官和行為學(xué)是否有影響,可能為重組人aB-晶體蛋白臨床應(yīng)用奠定基礎(chǔ),為臨床治療視神經(jīng)損傷提供新的途徑。 【目的】 建立重組蛋白表達(dá)體系,重組人αB-晶體蛋白和有更強(qiáng)分子伴侶活性的Trx-αB-crystallin融合蛋白;通過(guò)建立大鼠視神經(jīng)鉗夾傷模型,進(jìn)一步明確重組人αB-晶體蛋白對(duì)視神經(jīng)損傷后視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的保護(hù)作用;在此基礎(chǔ)上,通過(guò)動(dòng)物模型電生理檢查分析重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白治療視神經(jīng)損傷的療效,并通過(guò)顯微組織學(xué)檢查明確靜脈應(yīng)用重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白對(duì)全身重要臟器的影響,為開發(fā)重組人αB-晶體蛋白藥物治療視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷和視神經(jīng)損傷奠定實(shí)驗(yàn)基礎(chǔ)。 【方法】 1、根據(jù)已知人αB-晶體蛋白基因序列構(gòu)建目的基因,并建立目的基因載體質(zhì)粒pET32a和pET28a,通過(guò)BL21大腸桿菌細(xì)胞為宿主的表達(dá)系統(tǒng),IPTG誘導(dǎo)表達(dá)后純化,得到高純度的重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白,通過(guò)免疫印跡和蛋白質(zhì)譜分析鑒定重組蛋白。并利用小分子熱休克蛋白胰島素活性實(shí)驗(yàn),驗(yàn)證重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白的體外活性,同時(shí),通過(guò)對(duì)比研究觀察兩者分子伴侶活性強(qiáng)弱。 2、以成年Long Evans大鼠為研究對(duì)象,建立視神經(jīng)鉗夾損傷模型,靜脈注射重組人αB-晶體蛋白、Trx-αB-crystallin融合蛋白、Trx蛋白和生理鹽水。通過(guò)大腦上丘和外側(cè)膝狀體熒光金逆行標(biāo)記和視網(wǎng)膜鋪片的免疫組化染色方法,分別于損傷后2周和4周觀察視網(wǎng)膜內(nèi)存留有活性的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞,并定量分析和比較各組視網(wǎng)膜神經(jīng)節(jié)細(xì)胞數(shù)量。 3、以成年Long Evans大鼠建立視神經(jīng)鉗夾損傷模型,靜脈注射重組人αB-晶體蛋白、Trx-αB-crystallin融合蛋白、Trx蛋白和生理鹽水。利用閃光視覺誘發(fā)電位檢測(cè)方法,于損傷后2、4周觀察各組大鼠視功能情況,探討重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白對(duì)視神經(jīng)損傷后視功能修復(fù)的影響。 4、利用行為學(xué)方法,觀察上述各組實(shí)驗(yàn)動(dòng)物于損傷后2、4周的行為學(xué)變化,并在4周時(shí)處死大鼠,取心、肝、脾、腎重要臟器行組織病理學(xué)檢查,評(píng)估靜脈應(yīng)用重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白的安全性。 【結(jié)果】 1、構(gòu)建的載體質(zhì)粒pET32a-αB-crystallin和pET28a-αB-crystallin,通過(guò)BL21大腸桿菌細(xì)胞表達(dá)和純化可以得到高純度的重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白。 2、重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白均具有分子伴侶活性,且Trx-αB-crystallin融合蛋白的活性大于重組人αB-晶體蛋白。 3、視神經(jīng)鉗夾傷后熒光金逆行標(biāo)記示,靜脈注射重組人αB-晶體蛋白組和Trx-αB-crystallin融合蛋白組在損傷后2周和4周與對(duì)照組相比均有較多的具有軸漿運(yùn)輸功能的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞存活,統(tǒng)計(jì)學(xué)差異顯著(P0.05)。 4、視網(wǎng)膜神經(jīng)節(jié)細(xì)胞免疫組化熒光染色示,視神經(jīng)鉗夾傷后2周和4周靜脈注射重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白組較Trx蛋白和生理鹽水組有更多的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞存活,統(tǒng)計(jì)學(xué)差異顯著(P0.05)。 5、視神經(jīng)鉗夾傷后2周脈注射重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白組大鼠較Trx蛋白和生理鹽水組視功能檢查(閃光視覺誘發(fā)電位)示P1波波幅較大,差異顯著(P0.05)。但4周時(shí),各組間P1波波幅和潛時(shí)差異不顯著。 6、大鼠視神經(jīng)鉗夾傷后4周,重組人αB-晶體蛋白、Trx-αB-crystallin融合蛋白組大鼠、Trx蛋白和生理鹽水組行為學(xué)檢測(cè)無(wú)顯著性差異,且各組重要臟器組織學(xué)檢查未見明顯異常。 【結(jié)論】 1、大腸桿菌表達(dá)體系可以成功表達(dá)有分子伴侶活性的重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白。 2、重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白在視神經(jīng)損傷后修復(fù)中發(fā)揮重要作用。通過(guò)靜脈多次給藥,可明顯減少視神經(jīng)損傷引起的視網(wǎng)膜節(jié)細(xì)胞死亡,保存更多有軸漿運(yùn)輸功能的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞,提示重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白有促進(jìn)視神經(jīng)損傷修復(fù)的作用,并可以在損傷早期有效保護(hù)視功能。 3、全身靜脈應(yīng)用重組人αB-晶體蛋白和Trx-αB-crystallin融合蛋白安全,不會(huì)引起重要臟器病變和行為學(xué)改變。 4、Trx-αB-crystallin融合蛋白中Trx標(biāo)簽可以增強(qiáng)αB-晶體蛋白的分子伴侶活性,但不能提高αB-晶體蛋白保護(hù)功能,提示αB-晶體蛋白的分子伴侶活性和保護(hù)細(xì)胞功能可能不完全是一個(gè)功能結(jié)構(gòu)域。
[Abstract]:In recent years , it has been shown that 偽 - B - crystallin is the most active small molecule protein in the family of optic nerve .

Purpose of the project

The expression system of recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein with stronger molecular partner activity were established . The effect of recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein on optic nerve injury was further clarified by establishing the rat optic nerve clamp injury model . The effects of recombinant human 偽 - B - crystallin and Trx - 偽B - crystallin fusion protein on the whole body organ were analyzed by electrophysiological examination of animal model .

Methodology

1 . The recombinant human 偽B - crystallin and Trx - 偽B - allallin fusion protein were induced by IPTG . The recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein were purified by IPTG . The recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein were identified by Western blot and protein analysis .

2 . Using adult Long Evans rats as the research object , the optic nerve clamp injury model was established , the recombinant human 偽B - crystallin , Trx - 偽B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The retinal ganglion cells were observed in the retinal memory at 2 and 4 weeks after injury , and the number of retinal ganglion cells in each group was analyzed and compared .

3 . A model of optic nerve clamp injury was established in adult Long Evans rats . The recombinant human 偽B - crystallin , Trx - 偽B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The effects of recombinant human 偽 - B - crystallin and Trx - 偽B - crystallin fusion protein on the restoration of optic nerve injury were investigated in 2 and 4 weeks after injury .

4 . Using the behavioral method , the behavioral changes of 2 and 4 weeks after injury were observed and the safety of the recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein was assessed by histopathological examination of the rat , heart , liver , spleen and kidney in 4 weeks .

The result is not valid .

1 . The recombinant human 偽B - crystallin and Trx - 偽B - allallin fusion protein can be obtained by the expression and purification of E . coli cells .

2 . The recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein all have molecular partner activity , and the activity of Trx - 偽B - crystallin fusion protein is greater than that of recombinant human 偽B - crystallin .

3 . Compared with the control group , the recombinant human 偽B - crystallin group and Trx - 偽B - crystallin fusion protein group had more retinal ganglion cell survival than the control group at 2 and 4 weeks after injury , and the statistical difference was significant ( P0.05 ) .

4 . Immunohistochemical staining of retinal ganglion cells showed that the recombinant human 偽 - B - crystallin and Trx - 偽B - crystallin fusion protein group had more retinal ganglion cells survival after 2 and 4 weeks after the optic nerve clamp injury , and the statistical difference was significant ( P0.05 ) .

5 . After optic nerve clamp injury 2 weeks after injury , the recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein group showed a larger amplitude of P1 wave than that of Trx protein and physiological saline group ( P0.05 ) . But at 4 weeks , there was no significant difference between P1 wave amplitude and latent time difference among the groups .

6 . There was no significant difference in the behavior of recombinant human 偽B - crystallin , Trx - 偽B - allallin fusion protein group , Trx protein and physiological saline group at 4 weeks after optic nerve clamp injury in rats , and no significant abnormality was seen in histological examination of major organs in each group .

Conclusion

1 . The recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein expressed by the expression system of E . coli were successfully expressed .

2 . The recombinant human 偽B - crystallin and Trx - 偽B - allallin fusion protein play an important role in the repair of optic nerve injury . The retinal ganglion cells caused by optic nerve injury can be significantly reduced by intravenous administration of multiple doses , and more retinal ganglion cells with axonal transport function are preserved . It is suggested that the recombinant human 偽B - crystallin and Trx - 偽B - crystallin fusion protein have the effect of promoting the repair of optic nerve injury , and can effectively protect the visual function during early injury .

3 . The safety of recombinant human 偽 - B - crystallin and Trx - 偽B - crystallin fusion protein in systemic vein will not cause major organ changes and behavioral changes .

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本文編號(hào)：2135232