病毒样颗粒型结缔组织生长因子疫苗抑制小鼠四氯化碳诱导

StephenW

A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice

• 
  Shuang Li
• , Yi-Fei Lv
• , Hou-Qiang Su
• , Qian-Nan Zhang
• , Li-Rong Wang
• & Zhi-Ming Hao
  

• Scientific Reports 6, Article number: 32155 (2016)
• doi:10.1038/srep32155
• Download Citation
• 
  
  • Genetic engineering
  • Liver fibrosis
  • Target validation
    

Received:29 June 2015Accepted:03 August 2016Published online:26 August 2016


AbstractConnective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138–159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1–149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased α-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-β1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.


IntroductionLiver fibrosis, a common pathological process in a broad spectrum of chronic liver diseases, is characterized by the accumulation of extracellular matrix (ECM) resulting from the activation and/or epithelial-to-mesenchymal transition (EMT) of liver cells such as hepatic stellate cells (HSCs), portal fibroblasts and potentially hepatocytes into ECM-producing cells. The transformation of the ECM-producing cells is dependent on a number of extracellular stimuli such as cytokines, growth factors, chemokines, integrins and cell-cell interactions1. In the last decade, connective tissue growth factor (CTGF, or CCN2) has been identified as a central mediator in tissue fibrosis, including hepatic fibrosis2,3.
CTGF/CCN2 is a 38-kDa multifunctional secretory protein that is involved in multiple cellular events such as cell survival, proliferation, differentiation, migration, adhesion, angiogenesis, skeletal development and tissue repair, and oncogenesis, and is critically involved in tissue fibrosis4,5. CTGF is produced by most major cell types in the liver, especially activated HSCs, in response to diverse stimuli and is up-regulated in fibrotic livers at both the mRNA and protein levels6,7. The transgenic expression of CTGF in mouse hepatocytes in vivo renders the livers more susceptible to the injurious actions of other fibrotic stimuli8. Additionally and perhaps more convincingly, inhibiting the expression of CTGF9,10,11,12 or blocking its biological activity13,14 ameliorates experimental hepatic fibrosis. Moreover, the profibrogenic role of CTGF in other tissues and organs has been verified5. Therefore, CTGF is considered to be a fibrogenic master switch and a potential therapeutic target for hepatic fibrosis.
Vaccines against pathological cytokines, growth factors and extracellular soluble proteins have been proposed as a novel class of therapeutics and have been investigated in a number of disease models and clinical trials15,16,17,18,19,20,21,22,23,24,25,26,27,28. By cross-linking21, 26,27,28 or creating fusion proteins15,16,17,18,19,20,22,23,24,25,26 with carrier proteins, the normally non-antigenic cytokines or growth factors can be converted into self-antigens to elicit specific antibodies (Abs) through immunization; consequently, the Abs can neutralize abnormally overproduced cytokines or growth factors and inhibit their deleterious effects in pathological tissues. In the present study, we prepared a virus-like particle (VLP) CTGF vaccine by inserting a CTGF-derived polypeptide (aa 138–159) into the major immunodominant region (MIR) of the C-terminus truncated hepatitis B virus core antigen (HBc), tested its antigenicity and verified its protective effect in CCl4-induced hepatic fibrosis in BALB/c mice.

StephenW

病毒样颗粒型结缔组织生长因子疫苗抑制小鼠四氯化碳诱导的肝纤维化

    李爽，易飞吕侯强苏，钱楠张，李，王蓉与志明浩

    科学报告6，商品编号：32155（2016）
    DOI：10.1038 / srep32155
    下载文献
        遗传engineeringLiver fibrosisTarget验证

收稿日期：
    2015年6月29日
公认：
    2016年08月03日
网络发布时间：
    2016年8月26日

抽象

结缔组织生长因子（CTGF）已被确认为在肝纤维化的中心介体和有希望的治疗靶标。在这项研究中，我们通过将CTGF的138-159氨基酸（AA）片段插入到C末端截短的乙肝病毒核心抗原的中心C / E1的表位产生的新型病毒样颗粒（VLP）CTGF疫苗（HBC使用原核表达系统氨基酸1-149）。与VLP疫苗BALB / c小鼠的免疫有效诱发产生抗CTGF中和抗体。接种这CTGF疫苗从四氯化碳（四氯化碳）显著保护BALB / c小鼠诱导的肝纤维化，指示通过降低肝羟脯氨酸含量和较低的纤维化得分。四氯化碳中毒诱导的肝星状细胞的活化是由接种抑制，指示通过减小α平滑肌肌动蛋白的表达和Smad2的磷酸化。针对CTGF接种也减毒的一些促纤维化因子，如CTGF过度表达，在纤维化小鼠肝脏转化金属蛋白酶-1的生长因子β1，血小板衍生生长因子B和组织抑制剂，降低肝细胞凋亡和加速肝细胞增生纤维化小鼠肝脏。我们的结果清楚地表明，抗CTGF接种抑制纤维化，减轻肝细胞凋亡和促进肝再生。我们建议的疫苗应发展成肝纤维化的有效治疗手段。
介绍

肝纤维化，慢性肝脏疾病的广谱一个共同的病理过程，其特征在于细胞外基质（ECM）的积累从激活和/或上皮到间充质肝细胞的转化（EMT），导致如肝星状细胞（HSC），门静脉的成纤维细胞和潜在的肝细胞进入ECM产生细胞。对ECM产生细胞的转化是取决于许多细胞外刺激如细胞因子，生长因子，趋化因子，整合素和细胞 - 细胞interactions1的。在过去十年中，结缔组织生长因子（CTGF或CCN2）已被确定为在组织纤维化的中心介体，包括肝fibrosis2,3。

CTGF / CCN2]是参与多种细胞事件例如细胞存活，增殖，分化，迁移，粘附，血管生成，骨骼发育和组织修复，和肿瘤38 kDa的多功能分泌蛋白，并且是极其参与组织fibrosis4， 5。 CTGF是通过在肝最重要的细胞类型，尤其是活化的HSC中，响应于各种刺激产生和被上调在mRNA和蛋白levels6,7两个纤维化肝脏。 CTGF在小鼠体内肝细胞的转基因表达呈现肝脏其他纤维化stimuli8的有害行为更为敏感。此外，或许更具有说服力，抑制CTGF9,10,11,12的表达或阻断其生物activity13,14可改善实验性肝纤维化。此外，结缔组织生长因子在其它组织和器官的纤维化作用已经verified5。因此，CTGF被认为是一个纤维化主开关和肝纤维化的潜在的治疗靶标。

针对病理细胞因子，生长因子和细胞外可溶性蛋白的疫苗已被提议作为一类新的治疗剂，并在许多疾病模型和临床trials15,16,17,18,19,20,21,22,23已被研究， 24,25,26,27,28。通过交linking21，26,27,28或创建具有载体蛋白融合proteins15,16,17,18,19,20,22,23,24,25,26，常无抗原的细胞因子或生长因子可以被转换成自身抗原以引发通过免疫特异性抗体（ABS）;因此，在ABS可中和异常过度产生的细胞因子或生长因子，抑制在病变组织的有害作用。在本研究中，我们通过插入CTGF衍生的多肽（氨基酸138-159）插入主要免疫显性区的C末端截短的乙肝病毒核心抗原（MIR）（其制备的病毒样颗粒（VLP）疫苗CTGF的HBc），测试其抗原性和验证在BALB / c小鼠四氯化碳诱导的肝纤维化的保护作用。