TAT-Cytoglobin融合蛋白促進皮膚創(chuàng)傷愈合和抗肝纖維化初步研究
發(fā)布時間：2018-07-27 15:37
【摘要】：細胞珠蛋白（Cytoglobin，Cygb）是Kawada等在大鼠肝星狀細胞中發(fā)現(xiàn)的一種六配位血紅素球蛋白，分子量為21.4kDa。目前研究表明Cygb具有過氧化物酶活性和清除氧自由基的功能，并能通過調(diào)控Cygb基因的表達抑制肝纖維化。但Cygb作為一種胞漿蛋白，且在細胞表面無Cygb受體的情況下，外源Cygb無法主動進出細胞，發(fā)揮其抗氧化、抗炎癥及抗肝纖維化等生物學(xué)功能。本論文結(jié)合穿膜肽TAT的穿膜作用和Cygb的生物學(xué)功能，制備及探討TAT-Cygb融合蛋白在促進皮膚創(chuàng)傷愈合及抗肝纖維化方面的作用。 首先，通過乳糖誘導(dǎo)表達E. coli BL21/pET22b-TAT-Cygb中的TAT-Cygb融合蛋白，經(jīng)CM陽離子交換層析、S-100分子篩層析、G-25分子篩脫鹽等分離純化過程，以及SDS-PAGE、LC/MS和Western blot鑒定，得到純度95%、濃度為3.273mg/mL、過氧化物酶比活力為（422.63±3.17）U/mg的TAT-Cygb，并通過免疫細胞化學(xué)鑒定TAT-Cygb的穿膜能力；其次，利用H2O2氧化應(yīng)激損傷模型和超氧化物陰離子熒光探針證明TAT-Cygb對細胞的保護作用和治療氧化損傷的作用，并能降低細胞內(nèi)超氧化物陰離子的水平，而無穿膜能力的Cygb無法緩解已氧化損傷的細胞；再次，利用二甲苯致小鼠耳廓腫脹模型驗證TAT-Cygb的抗炎作用，在大鼠全層皮膚切除的創(chuàng)傷愈合實驗中，TAT-Cygb可發(fā)揮抗炎作用促進皮膚創(chuàng)傷愈合，并通過調(diào)節(jié)細胞內(nèi)的氧化應(yīng)激防止成纖維細胞過度增殖或遷移，從而有效減輕皮膚愈合后瘢痕修復(fù)區(qū)域的大�。蛔詈�，在抗小鼠肝纖維化實驗中，高劑量的TAT-Cygb可逆轉(zhuǎn)CCl4誘導(dǎo)的小鼠肝纖維化，，與模型組相比，肝臟內(nèi)膠原纖維含量明顯減少，肝小葉結(jié)構(gòu)恢復(fù)正常，肝功能指標和纖維化血清標志物等含量均趨于正常。 本論文首次利用TAT的穿膜能力介導(dǎo)Cygb進入胞內(nèi)，并證明TAT-Cygb融合蛋白具有促進皮膚創(chuàng)傷愈合及抗肝纖維化的作用，為后續(xù)開展Cygb抗炎癥、抗衰老、抗肝纖維化的應(yīng)用研究奠定了科學(xué)基礎(chǔ)。
[Abstract]:Cytoglobin (Cygb) is a hexagonal heme globulin found by Kawada et al in rat hepatic stellate cells with a molecular weight of 21.4 kDa. Current studies have shown that Cygb has the function of peroxidase activity and scavenging oxygen free radicals, and can inhibit hepatic fibrosis by regulating the expression of Cygb gene. However, as a cytoplasmic protein and without Cygb receptor on the cell surface, exogenous Cygb can not actively enter or leave the cells and play its biological functions such as antioxidation, anti-inflammation and anti-hepatic fibrosis. In this paper, the transmembrane effect of transmembrane peptide TAT and the biological function of Cygb were combined to prepare and investigate the role of TAT-Cygb fusion protein in promoting skin wound healing and anti-hepatic fibrosis. Firstly, the TAT-Cygb fusion protein of E. coli BL21/pET22b-TAT-Cygb was induced by lactose and purified by CM cation exchange chromatography, S-100 molecular sieve chromatography, G-25 molecular sieve desalination, and identified by SDS-PAGEL / MS and Western blot. TAT-Cygb with a purity of 95% and a concentration of 3.273 mg / mL with specific peroxidase activity of (422.63 鹵3.17) U/mg was obtained, and the membrane penetration ability of TAT-Cygb was identified by immunocytochemistry. H2O2 oxidative stress injury model and superoxide anion fluorescence probe were used to prove the protective effect of TAT-Cygb on cells and the effect of treating oxidative damage, and to reduce the level of superoxide anion in cells. However, Cygb without the ability to penetrate membrane could not alleviate the oxidative damage. Thirdly, the anti-inflammatory effect of TAT-Cygb was verified by using the model of auricle swelling induced by xylene in mice. TAT-Cygb may play an anti-inflammatory role in promoting skin wound healing in rats with full-thickness skin excision, and prevent fibroblast proliferation or migration by regulating intracellular oxidative stress. In the experiment of anti-liver fibrosis in mice, high dose of TAT-Cygb could reverse the hepatic fibrosis induced by CCl4, compared with the model group, the content of collagen fibers in liver decreased obviously. The structure of hepatic lobule returned to normal and the contents of liver function indexes and serum markers of fibrosis tended to be normal. In this paper, the transmembrane ability of TAT was used to mediate Cygb into the cell for the first time, and it was proved that the TAT-Cygb fusion protein could promote the healing of skin trauma and the anti-fibrosis of liver, and it could be used to carry out Cygb anti-inflammation and anti-aging. The application of anti-hepatic fibrosis has laid a scientific foundation.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965
本文編號：2148294
[Abstract]:Cytoglobin (Cygb) is a hexagonal heme globulin found by Kawada et al in rat hepatic stellate cells with a molecular weight of 21.4 kDa. Current studies have shown that Cygb has the function of peroxidase activity and scavenging oxygen free radicals, and can inhibit hepatic fibrosis by regulating the expression of Cygb gene. However, as a cytoplasmic protein and without Cygb receptor on the cell surface, exogenous Cygb can not actively enter or leave the cells and play its biological functions such as antioxidation, anti-inflammation and anti-hepatic fibrosis. In this paper, the transmembrane effect of transmembrane peptide TAT and the biological function of Cygb were combined to prepare and investigate the role of TAT-Cygb fusion protein in promoting skin wound healing and anti-hepatic fibrosis. Firstly, the TAT-Cygb fusion protein of E. coli BL21/pET22b-TAT-Cygb was induced by lactose and purified by CM cation exchange chromatography, S-100 molecular sieve chromatography, G-25 molecular sieve desalination, and identified by SDS-PAGEL / MS and Western blot. TAT-Cygb with a purity of 95% and a concentration of 3.273 mg / mL with specific peroxidase activity of (422.63 鹵3.17) U/mg was obtained, and the membrane penetration ability of TAT-Cygb was identified by immunocytochemistry. H2O2 oxidative stress injury model and superoxide anion fluorescence probe were used to prove the protective effect of TAT-Cygb on cells and the effect of treating oxidative damage, and to reduce the level of superoxide anion in cells. However, Cygb without the ability to penetrate membrane could not alleviate the oxidative damage. Thirdly, the anti-inflammatory effect of TAT-Cygb was verified by using the model of auricle swelling induced by xylene in mice. TAT-Cygb may play an anti-inflammatory role in promoting skin wound healing in rats with full-thickness skin excision, and prevent fibroblast proliferation or migration by regulating intracellular oxidative stress. In the experiment of anti-liver fibrosis in mice, high dose of TAT-Cygb could reverse the hepatic fibrosis induced by CCl4, compared with the model group, the content of collagen fibers in liver decreased obviously. The structure of hepatic lobule returned to normal and the contents of liver function indexes and serum markers of fibrosis tended to be normal. In this paper, the transmembrane ability of TAT was used to mediate Cygb into the cell for the first time, and it was proved that the TAT-Cygb fusion protein could promote the healing of skin trauma and the anti-fibrosis of liver, and it could be used to carry out Cygb anti-inflammation and anti-aging. The application of anti-hepatic fibrosis has laid a scientific foundation.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965
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