TAT-Cytoglobin融合蛋白促进皮肤创伤愈合和抗肝纤维化初步研究

发布时间：2018-07-27 15:37

【摘要】：细胞珠蛋白（Cytoglobin，Cygb）是Kawada等在大鼠肝星状细胞中发现的一种六配位血红素球蛋白，分子量为21.4kDa。目前研究表明Cygb具有过氧化物酶活性和清除氧自由基的功能，并能通过调控Cygb基因的表达抑制肝纤维化。但Cygb作为一种胞浆蛋白，且在细胞表面无Cygb受体的情况下，外源Cygb无法主动进出细胞，发挥其抗氧化、抗炎症及抗肝纤维化等生物学功能。本论文结合穿膜肽TAT的穿膜作用和Cygb的生物学功能，制备及探讨TAT-Cygb融合蛋白在促进皮肤创伤愈合及抗肝纤维化方面的作用。首先，通过乳糖诱导表达E. coli BL21/pET22b-TAT-Cygb中的TAT-Cygb融合蛋白，经CM阳离子交换层析、S-100分子筛层析、G-25分子筛脱盐等分离纯化过程，以及SDS-PAGE、LC/MS和Western blot鉴定，得到纯度95%、浓度为3.273mg/mL、过氧化物酶比活力为（422.63±3.17）U/mg的TAT-Cygb，并通过免疫细胞化学鉴定TAT-Cygb的穿膜能力；其次，利用H2O2氧化应激损伤模型和超氧化物阴离子荧光探针证明TAT-Cygb对细胞的保护作用和治疗氧化损伤的作用，并能降低细胞内超氧化物阴离子的水平，而无穿膜能力的Cygb无法缓解已氧化损伤的细胞；再次，利用二甲苯致小鼠耳廓肿胀模型验证TAT-Cygb的抗炎作用，在大鼠全层皮肤切除的创伤愈合实验中，TAT-Cygb可发挥抗炎作用促进皮肤创伤愈合，并通过调节细胞内的氧化应激防止成纤维细胞过度增殖或迁移，从而有效减轻皮肤愈合后瘢痕修复区域的大小；最后，在抗小鼠肝纤维化实验中，高剂量的TAT-Cygb可逆转CCl4诱导的小鼠肝纤维化，，与模型组相比，肝脏内胶原纤维含量明显减少，肝小叶结构恢复正常，肝功能指标和纤维化血清标志物等含量均趋于正常。本论文首次利用TAT的穿膜能力介导Cygb进入胞内，并证明TAT-Cygb融合蛋白具有促进皮肤创伤愈合及抗肝纤维化的作用，为后续开展Cygb抗炎症、抗衰老、抗肝纤维化的应用研究奠定了科学基础。
[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 卤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.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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