突变型人HGF(tvNK1)对CCl<sub>4</sub>诱导的大鼠肝纤维化的影响

doi:10.13523/j.cb.20160603

中国生物工程杂志

2016, Vol. 36

Issue (6): 18-23 DOI: 10.13523/j.cb.20160603

研究报告

突变型人HGF(tvNK1)对CCl₄诱导的大鼠肝纤维化的影响

王小花^1,2, 李玉婷¹, 刘亚威¹, 桂金秋¹, 周晓杭¹, 袁晓环², 初彦辉², 刘海峰²

1. 牡丹江医学院牡丹江医学院病原微生物与免疫实验室牡丹江 157011;
2. 牡丹江医学院黑龙江省抗纤维化重点实验室牡丹江 157011

Effects of Human Truncated Variant of Hepatocyte Growth Factor (tvNK1) on Carbon Tetrachloride-induced Liver Fibrosis in Rat

WANG Xiao-hua^1,2, LI Yu-ting¹, LIU Ya-wei¹, GUI Jin-qiu¹, ZHOU Xiao-hang¹, YUAN Xiao-huan², CHU Yan-hui², LIU Hai-feng²

1. Laboratory of Pathogenic Microbiology and Immunology, Mudanjiang Medical University, Mudanjiang 157011, China;
2. Heilongjiang Province Key Laboratory for Anti-Fibrosis Biotherapy, Mudanjiang Medical University, Mudanjiang 157011, China

全文: PDF(899 KB) HTML

摘要：

目的:探讨突变型人肝细胞生长因子(HGF^K132E,R134E, tvNK1)对四氯化碳(CCl₄)诱导的SD大鼠肝纤维化的影响。方法:生物发酵大量制备tvNK1,并经腹腔注射于CCl₄诱导的纤维化SD大鼠体内,6周后取肝脏组织,通过real-time PCR和Western blot检测tvNK1对纤维化SD大鼠肝脏中 I 型胶原蛋白(Collagen type I, Col1A1)、IV 型胶原蛋白(Collagen type IV, Col4A1)和 α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)在mRNA和蛋白水平表达的影响,并进一步通过HE和Masson染色观察tvNK1对纤维化SD大鼠肝脏形态和肝组织胶原纤维的影响。结果:SDS-PAGE检测结果显示,获得纯度≥95%的tvNK1。Real-time PCR和Western blot结果显示,tvNK1降低纤维化大鼠肝脏中Col1A1、Col4A1 和 α-SMA在mRNA和蛋白水平上的表达。HE和Masson染色结果显示,tvNK1减缓纤维化大鼠肝结构的病理性改变,并降低肝脏中胶原纤维含量。结论:tvNK1能抑制CCl₄诱导的大鼠肝纤维化,为预防肝纤维化和其他器官纤维化疾病提供实验依据。

关键词： 突变型人肝细胞生长因子; 四氯化碳; 肝纤维化; 胶原纤维

Abstract:

Objective: In order to determine the effects of human truncated variant of hepatocyte growth factor (tvNK1) on carbon tetrachloride (CCl₄)-induced liver fibrosis in SD rat. Methods: The recombinant tvNK1 was high-level expression in Escherichia coli BL21 by bio-fermentation and rapid purification of tvNK1 protein using Ni²⁺-affinity chromatograph. The SD rat model of CCl₄-induced hepatic fibrosis was received intraperitoneal injection of tvNK1 for 6 weeks. The mRNA and protein expression levels of Col1A1、Col4A1 and α-SMA in hepatic tissue of different groups were assayed by real-time PCR and Western blot, respectively. The morphology and the collagen fiber of liver sections in different groups were determined by Hematoxylin and eosin (HE) and Masson staining, respectively. Results: The high purity tvNK1 was assayed by SDS-PAGE. The recombinant tvNK1 reduced the mRNA and protein expression levels of Col1A1、Col4A1 and α-SMA in CCl₄-induced hepatic tissue in Rat. Moreover, injection with tvNK1 restored the morphology and the collagen fiber in CCl₄-induced hepatic tissue in Rat. Conclusion: The recombinant tvNK1 protein attenuated progression of hepatic fibrosis in rat and provided a potential treatment for liver fibrosis and other fibrosis diseases.

Key words: Collagen fiber Liver fibrosis Carbon tetrachloride (CCl₄) Truncated variant of hepatocyte growth factor (tvNK1)

收稿日期: 2015-12-18 出版日期: 2016-03-02

ZTFLH:

Q819

基金资助:

国家自然科学基金(81573068,81500471)、黑龙江省自然科学基金(H2015080)、黑龙江省组织损伤与修复重点实验室开放课题(zdsys2013-07,zdsys2013-03)资助项目

通讯作者: 刘海峰, 初彦辉 E-mail: fenghailiu510@163.com;yanhui_chu@sina.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王小花
	李玉婷
	刘亚威
	桂金秋
	周晓杭
	袁晓环
	初彦辉
	刘海峰

引用本文:

王小花, 李玉婷, 刘亚威, 桂金秋, 周晓杭, 袁晓环, 初彦辉, 刘海峰. 突变型人HGF(tvNK1)对CCl₄诱导的大鼠肝纤维化的影响[J]. 中国生物工程杂志, 2016, 36(6): 18-23.

WANG Xiao-hua, LI Yu-ting, LIU Ya-wei, GUI Jin-qiu, ZHOU Xiao-hang, YUAN Xiao-huan, CHU Yan-hui, LIU Hai-feng. Effects of Human Truncated Variant of Hepatocyte Growth Factor (tvNK1) on Carbon Tetrachloride-induced Liver Fibrosis in Rat. China Biotechnology, 2016, 36(6): 18-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160603 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/18

[1] Ghazwani M, Zhang Y, Gao X, et al. Anti-fibrotic effect of thymoquinone on hepatic stellate cells. Phytomedicine,2014,21(3):254-260.
[2] Neaud V, Faouzi S, Guirouilh J, et al. Human hepatic myofibroblasts increase invasiveness of hepatocellular carcinoma cells: evidence for a role of hepatocyte growth factor. Hepatology,1997,26(6):1458-1466.
[3] 晏维, 唐望先. 肝细胞生长因子与肝纤维化的研究进展. 国际消化病杂志,2007, 2(1):12-15. Yan W, Tang W X. Research progression of hepatocyte growth factor and liver fibrosis. Int J Dig Dis,2007, 2(1):12-15.
[4] Patijn G A, Lieber A, Schowalter D B, et al. Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice. Hepatology,1998,28(3):707-716.
[5] Xue F, Takahara T, Yata Y, et al. Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy. Gut,2003,52(5):694-700.
[6] Wang X, Liu H, Zhang Z, et al. High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli. World J Microbiol Biotechnol,2014,30(11):2851-2859.
[7] 张西轩, 李晔, 王亚航, 等. 蜡样芽孢杆菌ColR75E 胶原酶的表达、纯化及酶学性质研究.中国生物工程杂志,2015,35(10):44-52. Zhang X X, Li Y, Wang Y H, et al. Expression, purification and enzymatic characterization of ColR75E collagenase of Bacillus cereus R75E. China Biotechnology,2015, 35(10):44-52.
[8] Liu H, Wang X, Wang C, et al. Molecular cloning, in vitro expression and bioactivity of rabbit transforming growth factor-beta receptor type II (rTGF-betaRII). Vet Immunol Immunopathol,2011,140(1-2):55-62.
[9] Sanchez-Valle V, Chavez-Tapia N C, Uribe M, et al. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem,2012,19(28):4850-4860.
[10] 李明月, 刘立忠, 黄尚志, 等。肝细胞生长因子抑制MRC-5成纤维细胞所致的纤维化作用.基础医学与临床,2002,22(5):422-427. Li M Y, Liu L Z, Huang S Z, et al. Hepatocyte growth factor suppress fibrogenic events in myofibroblastic MRC-5. Basic & Clinical Medicine,2002,22(5):422-427.
[11] Masunaga H, Fujise N, Shiota A, et al. Preventive effects of the deleted form of hepatocyte growth factor against various liver injuries. Eur J Pharmacol,1998,342 (2-3):267-279.
[12] Matsuda Y, Matsumoto K, Yamada A, et al. Preventive and therapeutic effects in rats of hepatocyte growth factor infusion on liver fibrosis/cirrhosis. Hepatology,1997,26(1):81-89.
[13] Nakamura T, Sakata R, Ueno T, et al. Inhibition of transforming growth factor beta prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology,2000,32(2):247-255.
[14] 周伟,沈薇.肝细胞生长因子对肝纤维化的保护作用及其机制研究. 重庆医学,2009, 38(4):398-402. Zhou W, Shen W. Protective effects of HGF on experimental liver fibrosis in rats and analysis of its mechanisms. Chongqing Yixue,2009,38(4):398-402.
[15] 孙瑞芬,张艳芳,郭树春,等.向日葵ACC 氧化酶基因(HaACO1)的克隆及表达分析.中国生物工程杂志,2015,35(9):21-27. Sun R F, Zhang Y F, Guo S C, et al. Cloning and expression analysis of ACC oxidase gene ( HaACO1 ) from sunflower (Helianthus annuus L.). China Biotechnology,2015,35(9): 21-27.
[16] Mars W M, Liu M L, Kitson R P, et al. Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration. Hepatology,1995,21(6):1695-1701.
[17] Ross J, Gherardi E, Mallorqui-Fernandez N, et al. Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver. Gastroenterology,2012,142(4):897-906.
[18] Gherardi E, Sandin S, Petoukhov M V, et al. Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc Natl Acad Sci U S A,2006,103(11):4046-4051.

[1]	丛姗, 王秀梅, 李岩, 宋瑾, 白立恒, 曹贵方. 人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤定位修复的研究[J]. 中国生物工程杂志, 2014, 34(8): 14-23.
[2]	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.

Viewed

Full text

Abstract

Cited

Shared

Discussed