重组截短型TGF-βⅡ型受体蛋白对肝纤维化大鼠的治疗作用研究

doi:10.13523/j.cb.20141203

中国生物工程杂志

2014, Vol. 34

Issue (12): 16-22 DOI: 10.13523/j.cb.20141203

研究报告

重组截短型TGF-βⅡ型受体蛋白对肝纤维化大鼠的治疗作用研究

马洪闯^1,2, 柏合², 张哲², 赵冰海², 曹雅男², 李洪志², 刘洁婷², 张春雷², 冯彪³, 初彦辉^1,2,3

1. 佳木斯大学基础医学院生物化学与分子生物学系佳木斯 154007;
2. 牡丹江医学院黑龙江省抗纤维化生物治疗重点实验室牡丹江 157011;
3. 牡丹江医学院黑龙江省高校组织损伤与修复重点实验室牡丹江 157011

Therapeutically Effect of a Novel Truncated Transforming Growth Factor-β Type Ⅱ Receptor Protein in Rat Hepatic Fibrosis

MA Hong-chuang^1,2, BAI He², ZHANG Zhe², ZHAO Bing-hai², CAO Ya-nan², LI Hong-zhi², LIU Jie-ting², ZHANG Chun-lei², FENG Biao³, CHU Yan-hui^1,2,3

1. Department of Biochemistry and Molecular Biology, Jiamusi University, Jiamusi 154007, China;
2. Key Laboratory of Heilongjiang for Anti-fibrosis Biotherapy, Mudanjiang Medical University, Mudanjiang 157011, China;
3. Key Laboratory of Heilongjiang for Tissue Damage and Repair, Mudanjiang Medical University, Mudanjiang 157011, China

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摘要：

目的:研究截短型TGF-βⅡ型受体蛋白对肝纤维化大鼠的治疗作用。方法:取含有重组截短型TGF-βⅡ型受体载体的大肠杆菌BL21,通过培养繁殖,诱导,纯化得到重组截短型TGF-βⅡ型受体蛋白。使用CCl₄诱导的方法来制备肝纤维化大鼠模型,观察截短型TGF-βⅡ型受体蛋白对大鼠肝纤维化的影响。SDS-PAGE检测纯化的截短型TGF-βⅡ型受体蛋白。生化检测血清谷丙转氨酶(glutamic pyruvic transaminase,ALT)及谷草转氨酶(glutamic oxalacetic transaminase,AST)含量。用实时荧光定量PCR(real-time PCR)方法和免疫荧光方法来检测各组大鼠肝组织中的α平滑肌肌动蛋白(alpha-smooth muscle actin, α-SMA),1型胶原(collagen1, Col1)和4型胶原(collagen4, Col4)的表达情况。用HE,MASSON,PAS这三种病理学染色的方法来观察各组大鼠肝纤维化的变化。结果:SDS-PAGE检测结果显示,纯化后获得高纯度截断型TGF-βⅡ型受体蛋白。截短型TGF-βⅡ型受体蛋白治疗组可以减少血清中谷丙转氨酶和谷草转氨酶的含量。截短型TGF-βⅡ型受体蛋白治疗组可以降低肝组织中α-SMA、FN、Col1和Col4的mRNA和蛋白质的表达。三种病理学染色的方法显示,与肝纤维化大鼠模型组相比,治疗组的纤维化程度明显减轻。结论:重组截短型TGF-βⅡ型受体蛋白对大鼠肝纤维化的程度有抑制作用,为治疗肝纤维化及其他纤维化疾病提供了一个潜在的治疗手段。

关键词： 肝纤维化; 转化生长因子β 受体

Abstract:

Objective: Examine the anti-fibrotic effect of a novel truncated transforming growth factor-β typeⅡ receptor (tTGF-βRⅡ) in rat hepatic fibrosis model.Methods: The recombinant truncated type TGF-betaⅡ receptor proteins was obtained from the Escherichia coli BL21 strain containing recombinant truncated TGF-betaⅡ receptor vector, by cultivating, inducing, and purification. The rat model of CCL₄-induced hepatic fibrosis was established to assess the effect of the tTGF-βRⅡ protein on the treatment of fibrosis. SDS-PAGE detect the purification of truncated TGF-betaⅡ receptor proteins. The mRNA expression levels of α-SMA,Col1 and Col4 were detected by real-time PCR analysis in hepatic tissue. The content of serum ALT and AST were checked with automated biochemistry analyzer. The expression of α-SMA,Col1 and Col4 protein in hepatic tissue was detected by immunofluorescence. Liver fibrosis of the rats was evaluated by three histological methods: HE staining,Masson staining and PAS staining. Results: SDS-PAGE detection results show that the purified recombinant truncated TGF-beta Ⅱ receptor proteins was obtained. Histological examination revealed that the tTGF-βRⅡ protein prevented progression of hepatic fibrosis. The tTGF-βRⅡ protein reduced activities of serum ALT and AST. The tTGF-βRⅡ protein reduced both mRNA and protein expression of α-SMA,Col1 and Col4 in the liver tissue. The results of three histopathological staining shows that the treatment group significantly reduce the degree of liver fibrosis.Conclusion: The tTGF-βRⅡ protein prevented progression of hepatic fibrosis in rat and provided a potential treatment for liver fibrosis and other fibrosis diseases.

Key words: Liver fibrosis Transforming growth factor-β Receptor

收稿日期: 2014-09-24 出版日期: 2014-12-25

ZTFLH:

Q819

基金资助:

国家自然科学基金青年科学基金(81200305)、2011年度黑龙江省教育厅重点研究项目计划(12511z028)资助项目

通讯作者: 初彦辉 E-mail: yanhui_chu@sina.com

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引用本文:

马洪闯, 柏合, 张哲, 赵冰海, 曹雅男, 李洪志, 刘洁婷, 张春雷, 冯彪, 初彦辉. 重组截短型TGF-βⅡ型受体蛋白对肝纤维化大鼠的治疗作用研究[J]. 中国生物工程杂志, 2014, 34(12): 16-22.

MA Hong-chuang, BAI He, ZHANG Zhe, ZHAO Bing-hai, CAO Ya-nan, LI Hong-zhi, LIU Jie-ting, ZHANG Chun-lei, FENG Biao, CHU Yan-hui. Therapeutically Effect of a Novel Truncated Transforming Growth Factor-β Type Ⅱ Receptor Protein in Rat Hepatic Fibrosis. China Biotechnology, 2014, 34(12): 16-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141203 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I12/16

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