IFN-α在NK细胞抗小鼠肝纤维化作用中的机制

发布时间：2018-05-21 07:27

本文选题：肝纤维化 + 不同阶段　；参考：《福建医科大学》2015年硕士论文

【摘要】：研究背景和目的:活化的肝脏星状细胞(hepatic stellate cells HSC)是肝纤维化进程中的核心细胞,肝脏自然杀伤细胞(natrual kill cell NK)细胞介导的免疫反应是调控纤维化进程的重要因素之一,干扰素-α(interferon-αIFN-α)可免疫调控NK细胞功能。1.本实验通过建立不同阶段纤维化小鼠肝脏NK/HSC体外共培养体系,研究肝脏NK细胞和HSC细胞之间的相互作用机制,进一步阐明NK细胞抗纤维化的作用机制。2.通过IFN-α直接干预NK/HSC共培养体系,检测NK细胞的细胞毒性及分泌干扰素-γ(interferon-γIFN-γ)功能,阐明IFN-α抗纤维化的免疫学机制,为临床应用IFN-α提供实验室证据。研究方法:1.取8周龄雄性C57BL/6J小鼠,腹腔注射四氯化碳(carbon tetrachloride CCL4)建立早期阶段(2周组)和晚期阶段(8周组)肝纤维化小鼠模型。不同密度梯度法提取不同纤维化阶段小鼠HSC,磁珠分选法提取不同纤维化阶段小鼠肝脏NK细胞,建立各组NK/HSC共培养体系。2.在各组共培养体系中加入抗小鼠肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor-related apoptosis-inducing ligand TRAIL)抗体、以抗小鼠NK细胞组2成员D(natural killer group 2 member D NKG2D)抗体、抗小鼠IFN-γ抗体和抗小鼠转化因子β1(transforming growth factorβ1 TGF-β1)抗体,共培养24小时,LDH检测法检测不同纤维化阶段小鼠NK细胞的细胞毒性。ELISA检测不同阶段纤维化小鼠肝脏NK细胞的细胞分泌IFN-γ功能。3.IFN-α干预各组纤维化小鼠NK/HSC共培养体系及HSC独立培养组。LDH检测法检测不同纤维化阶段小鼠NK细胞的细胞毒性。ELISA法检测不同阶段纤维化小鼠肝脏NK细胞的细胞分泌功能。流式细胞术检测HSC凋亡率。结果:1.成功建立2周组(早期)、8周组(晚期)肝纤维化模型小鼠,并分离、培养各组小鼠肝脏HSC和NK细胞,建立2周组、8周组NK/HSC共培养体系,加入相应抗体干预后,共培养24小时取得上清液。2、检测各组共培养体系的NK细胞毒性及分泌IFN-γ水平,得到以下结果:1)2周组小鼠肝脏NK/HSC共培养体系NK细胞毒性及分泌IFN-γ水平高于8周组(29.72%±0.49%VS17.52%±0.60 p0.01;24.27±0.68pg/ml VS 13.48±0.37pg/ml p0.01);2)以抗小鼠TRAIL抗体、以抗小鼠NKG2D抗体、抗小鼠IFN-γ抗体干预2周组、8周组小鼠肝脏NK/HSC共培养体系,其NK细胞毒性分泌IFN-γ水平均低于无抗体干预组(p0.01);3)以抗小鼠TGF-β1抗体干预2周组、8周组NK/HSC共培养体系,NK细胞毒性及分泌IFN-γ水平均高于无抗体干预组(54.08%±2.76%VS29.72%±1.49%p0.01;29.41±0.21pg/ml VS24.3±0.81pg/ml p0.01),(49.81%±2.93%VS17.52%±1.61%p0.01;19.87±0.23pg/ml VS13.38±0.38pg/ml p0.01)。且TGF-β1抗体对8周组小鼠肝脏NK细胞毒性及分泌IFN-γ增加程度大于2组周(p0.01)。3.以IFN-α干预2周组、8周组小鼠肝脏NK/HSC共培养体系NK细胞毒性高于无IFN-α干预组(45.71%±0.87%VS29.66%±0.38%p0.01;20.97%±0.70%VS16.39%±0.40%;p0.01)且2周组NK细胞毒性增加程度大于8周组(P0.01);但是IFN-α对NK细胞分泌IFN-γ的促进作用仅见于2周组(27.57±0.19pg/ml VS24.27±0.68pg/ml p0.01),8周组干预前后无统计学差异(p=0.1760.05)。IFN-α干预可通过增强NK细胞功能提高2周组、8周组NK/HSC共培养体系HSC凋亡率(27.37%±0.03%VS22.72%±0.16%t=-188.237,22.26%±0.03%VS20.39%±0.27%t=11.893,p0.01),而对HSC无直接诱导凋亡作用(P0.05)。结论:NK细胞可通过NKG2D、TRAIL表面受体途径及分泌大量的IFN-γ抑制杀伤活化的HSC,发挥其抗抑制纤维化作用。在晚期纤维化阶段NK细胞功能被HSC分泌的TGF-β1抑制,使其对HSC的杀伤能力下降,肝纤维化持续发展。在肝纤维化早期,IFN-α通过增强NK细胞毒性及分泌IFN-γ能力,增强NK细胞的抗纤维化作用,而这种作用在纤维化晚期因TGF-β1的强抑制作用效果不显著。总结:IFN-α可能过增强NK细胞功能抑制早期肝纤维化进展。
[Abstract]:Background and purpose: hepatic stellate cells HSC is the core cell of liver fibrosis, and the immune response mediated by natrual kill cell NK cells (NATRUAL kill cell NK) cells is one of the important factors regulating the process of fibrosis, and the interferon alpha (interferon- alpha IFN- alpha) can be immune to the regulation of NK cell function. 1. in this experiment, the interaction mechanism of liver NK cells and HSC cells was studied through the establishment of NK/HSC co culture system in different stages of liver fibrosis in mice. The mechanism of anti fibrosis of NK cells was further elucidated,.2. was directly interfered with NK/HSC co culture system through IFN- alpha, and the cytotoxicity of NK cells and the secretion of interferon gamma (inter) were detected. Feron- gamma IFN- gamma function, elucidate the immunological mechanism of IFN- a anti fibrosis and provide laboratory evidence for the clinical application of IFN- alpha. 1. the 8 weeks male C57BL/6J mice were taken and the abdominal injection of carbon tetrachloride (carbon tetrachloride CCL4) was injected into the early stage (2 weeks group) and the late stage (8 weeks group) model of liver fibrosis. Different density gradient The mouse liver NK cells were extracted from different fibrosis stages by the extraction of HSC in different fibrosis stages, and the NK/HSC co culture system.2. was established in each group, and the anti tumor necrosis factor related apoptosis inducing ligand (Tumor necrosis factor-related apoptosis-inducing ligand TRAIL) antibody was added to the co culture system of each group, and the anti tumor necrosis factor related apoptosis inducing ligand (Tumor necrosis factor-related apoptosis-inducing ligand TRAIL) was added to the mice. The 2 members of the mouse NK cell group, D (natural killer group 2 member D NKG2D) antibody, anti mouse IFN- gamma antibody and anti mouse transforming factor beta 1 (transforming growth factor beta 1 beta 1) antibody, were cultured for 24 hours. The cell cells secreted IFN- gamma function.3.IFN- alpha in the NK/HSC co culture system and the.LDH detection method of the HSC independent culture group to detect the cytotoxic activity of the NK cells in the liver of the mice with different stages of fibrosis by the.ELISA method. The rate of HSC apoptosis was detected by flow cytometry. The results were as follows: 1. successful establishment of 2 weeks group (early), 8 weeks group (late) liver fibrosis model mice, and isolated, cultured mice liver HSC and NK cells, set up 2 weeks group, 8 weeks group NK/HSC co culture system, add the corresponding antibody dry prognosis, co culture 24 hours to obtain the supernatant.2, detect the NK cytotoxicity and the secretion of IFN- gamma level in the co culture system of each group. The following results were as follows: 1) the NK cytotoxicity and secretion of IFN- gamma in the NK/HSC co culture system of mice in the 2 week group were higher than those in the 8 week group (29.72% + 0.49%VS17.52% + 0.60 P0.01; 24.27 + 0.68pg/ml VS 13.48 + 0.37pg/ml P0.01); 2) against mice TRAIL antibody, anti mice NKG2D antibody, anti mouse IFN- gamma antibody intervention for 2 weeks, and 8 weeks group of mice liver tissue culture. The NK cytotoxic secretion of IFN- gamma was lower than that of non antibody intervention group (P0.01), 3) with anti mouse TGF- beta 1 antibody intervention for 2 weeks, and 8 weeks group NK/HSC co culture system, NK cytotoxicity and secretion of IFN- gamma water were higher than that of non antibody intervention group (54.08% + 2.76%VS29.72% + 1.49%p0.01; 29.41 + 0.21pg/ml VS24.3 + 0.81pg/ml), (49.81% + 2) .93%VS17.52% + 1.61%p0.01; 19.87 + 0.23pg/ml VS13.38 + 0.38pg/ml P0.01). The degree of NK cytotoxicity and secretion of IFN- gamma in the liver of mice with TGF- beta 1 increased more than 2 weeks (P0.01).3. with IFN- alpha intervention for 2 weeks. The 8 week group of mice liver cancer co culture system was higher than that without alpha intervention group (45.71% +. 0.38) %p0.01; 20.97% + 0.70%VS16.39% + 0.40%; P0.01) and the increase of NK cell toxicity in 2 weeks group was more than 8 weeks (P0.01), but the promotion effect of IFN- alpha on IFN- gamma secreted by NK cells was only in the 2 week group (27.57 + 0.19pg/ml VS24.27 + 0.68pg/ml P0.01). The 8 week group had no statistical difference before and after the intervention. The apoptosis rate of HSC (27.37% + 0.03%VS22.72% + 0.16%t=-188.237,22.26% + 0.03%VS20.39% + 0.27%t=11.893, P0.01) in the NK/HSC co culture system was increased in the group of 2 weeks, but there was no direct apoptosis effect on HSC (P0.05). Conclusion: NK cells can play its resistance through NKG2D, TRAIL surface receptor path and secreting a large number of inhibitors. Inhibition of fibrosis. The function of TGF- beta 1 secreted by HSC in the stage of advanced fibrosis is inhibited by TGF- beta 1, which reduces the killing ability of HSC and the liver fibrosis continues to develop. In the early stage of liver fibrosis, IFN- alpha enhanced the anti fibrinolysis of NK cells by enhancing NK cytotoxicity and secretion of IFN- gamma, and this effect was due to TGF in the late fibrosis. Conclusion: the strong inhibitory effect of beta 1 is not significant. Conclusion: IFN- alpha may enhance the function of NK cells and inhibit the progression of early hepatic fibrosis.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R575.2

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