本文選題：肝纖維化 + 不同階段�。� 參考：《福建醫(yī)科大學(xué)》2015年碩士論文

【摘要】：研究背景和目的:活化的肝臟星狀細(xì)胞(hepatic stellate cells HSC)是肝纖維化進(jìn)程中的核心細(xì)胞,肝臟自然殺傷細(xì)胞(natrual kill cell NK)細(xì)胞介導(dǎo)的免疫反應(yīng)是調(diào)控纖維化進(jìn)程的重要因素之一,干擾素-α(interferon-αIFN-α)可免疫調(diào)控NK細(xì)胞功能。1.本實(shí)驗(yàn)通過(guò)建立不同階段纖維化小鼠肝臟NK/HSC體外共培養(yǎng)體系,研究肝臟NK細(xì)胞和HSC細(xì)胞之間的相互作用機(jī)制,進(jìn)一步闡明NK細(xì)胞抗纖維化的作用機(jī)制。2.通過(guò)IFN-α直接干預(yù)NK/HSC共培養(yǎng)體系,檢測(cè)NK細(xì)胞的細(xì)胞毒性及分泌干擾素-γ(interferon-γIFN-γ)功能,闡明IFN-α抗纖維化的免疫學(xué)機(jī)制,為臨床應(yīng)用IFN-α提供實(shí)驗(yàn)室證據(jù)。研究方法:1.取8周齡雄性C57BL/6J小鼠,腹腔注射四氯化碳(carbon tetrachloride CCL4)建立早期階段(2周組)和晚期階段(8周組)肝纖維化小鼠模型。不同密度梯度法提取不同纖維化階段小鼠HSC,磁珠分選法提取不同纖維化階段小鼠肝臟NK細(xì)胞,建立各組NK/HSC共培養(yǎng)體系。2.在各組共培養(yǎng)體系中加入抗小鼠腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(Tumor necrosis factor-related apoptosis-inducing ligand TRAIL)抗體、以抗小鼠NK細(xì)胞組2成員D(natural killer group 2 member D NKG2D)抗體、抗小鼠IFN-γ抗體和抗小鼠轉(zhuǎn)化因子β1(transforming growth factorβ1 TGF-β1)抗體,共培養(yǎng)24小時(shí),LDH檢測(cè)法檢測(cè)不同纖維化階段小鼠NK細(xì)胞的細(xì)胞毒性。ELISA檢測(cè)不同階段纖維化小鼠肝臟NK細(xì)胞的細(xì)胞分泌IFN-γ功能。3.IFN-α干預(yù)各組纖維化小鼠NK/HSC共培養(yǎng)體系及HSC獨(dú)立培養(yǎng)組。LDH檢測(cè)法檢測(cè)不同纖維化階段小鼠NK細(xì)胞的細(xì)胞毒性。ELISA法檢測(cè)不同階段纖維化小鼠肝臟NK細(xì)胞的細(xì)胞分泌功能。流式細(xì)胞術(shù)檢測(cè)HSC凋亡率。結(jié)果:1.成功建立2周組(早期)、8周組(晚期)肝纖維化模型小鼠,并分離、培養(yǎng)各組小鼠肝臟HSC和NK細(xì)胞,建立2周組、8周組NK/HSC共培養(yǎng)體系,加入相應(yīng)抗體干預(yù)后,共培養(yǎng)24小時(shí)取得上清液。2、檢測(cè)各組共培養(yǎng)體系的NK細(xì)胞毒性及分泌IFN-γ水平,得到以下結(jié)果:1)2周組小鼠肝臟NK/HSC共培養(yǎng)體系NK細(xì)胞毒性及分泌IFN-γ水平高于8周組(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抗體、以抗小鼠NKG2D抗體、抗小鼠IFN-γ抗體干預(yù)2周組、8周組小鼠肝臟NK/HSC共培養(yǎng)體系,其NK細(xì)胞毒性分泌IFN-γ水平均低于無(wú)抗體干預(yù)組(p0.01);3)以抗小鼠TGF-β1抗體干預(yù)2周組、8周組NK/HSC共培養(yǎng)體系,NK細(xì)胞毒性及分泌IFN-γ水平均高于無(wú)抗體干預(yù)組(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抗體對(duì)8周組小鼠肝臟NK細(xì)胞毒性及分泌IFN-γ增加程度大于2組周(p0.01)。3.以IFN-α干預(yù)2周組、8周組小鼠肝臟NK/HSC共培養(yǎng)體系NK細(xì)胞毒性高于無(wú)IFN-α干預(yù)組(45.71%±0.87%VS29.66%±0.38%p0.01;20.97%±0.70%VS16.39%±0.40%;p0.01)且2周組NK細(xì)胞毒性增加程度大于8周組(P0.01);但是IFN-α對(duì)NK細(xì)胞分泌IFN-γ的促進(jìn)作用僅見(jiàn)于2周組(27.57±0.19pg/ml VS24.27±0.68pg/ml p0.01),8周組干預(yù)前后無(wú)統(tǒng)計(jì)學(xué)差異(p=0.1760.05)。IFN-α干預(yù)可通過(guò)增強(qiáng)NK細(xì)胞功能提高2周組、8周組NK/HSC共培養(yǎng)體系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),而對(duì)HSC無(wú)直接誘導(dǎo)凋亡作用(P0.05)。結(jié)論:NK細(xì)胞可通過(guò)NKG2D、TRAIL表面受體途徑及分泌大量的IFN-γ抑制殺傷活化的HSC,發(fā)揮其抗抑制纖維化作用。在晚期纖維化階段NK細(xì)胞功能被HSC分泌的TGF-β1抑制,使其對(duì)HSC的殺傷能力下降,肝纖維化持續(xù)發(fā)展。在肝纖維化早期,IFN-α通過(guò)增強(qiáng)NK細(xì)胞毒性及分泌IFN-γ能力,增強(qiáng)NK細(xì)胞的抗纖維化作用,而這種作用在纖維化晚期因TGF-β1的強(qiáng)抑制作用效果不顯著�？偨Y(jié):IFN-α可能過(guò)增強(qiáng)NK細(xì)胞功能抑制早期肝纖維化進(jìn)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R575.2

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