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硫酸酯酶基因及其N端活性片段抑制肝星状细胞激活的作用机制研究

发布时间:2018-06-08 06:33

  本文选题:肝纤维化 + 原代肝星状细胞 ; 参考:《第二军医大学》2014年硕士论文


【摘要】:【研究背景与研究目的】 肝纤维化的病理生理发展过程与肝脏微环境中的多种炎性介质和细胞因子密切相关,其中最主要的就是各种促进纤维化发生发展的生长因子,这些生长因子主要包括TGF-β1、PDGF、CTGF、FGF、VEGF等等,这些生长因子发挥作用都需要和细胞表面的生长因子受体结合,进而激活与受体耦合的细胞内信号通路。这些生长因子和细胞表面的生长因子受体结合时,大多还需要存在于细胞表面的硫酸乙酰肝素蛋白聚糖(HSPG)分子中的硫酸乙酰肝素(HS)作为共同配体或提供空间构象。而大量研究表明,Sulf-1基因编码的硫酸酯酶恰好可以使硫酸乙酰肝素蛋白聚糖分子中的硫酸乙酰肝素发生去硫酸化,进而使硫酸乙酰肝素蛋白聚糖分子失去为生长因子受体提供共同配体和提供空间构象的能力,从而阻断生长因子信号的传递。我们猜测,Sulf-1基因同样能阻断与促进肝纤维化发生发展有关的生长因子的信号传递,从而达到阻断肝纤维化进程的目的。目前对肝纤维化的研究结果显示,在肝纤维化进程中起关键作用的就是肝星状细胞。于是,我们首先分离并检测了大鼠原代肝星状细胞中大鼠Sulf-1基因的表达水平随原代肝星状细胞激活状态的变化,构建了人Sulf-1基因的腺病毒载体Ad5-hSulf1,人Sulf-1基因的沉默载体hSulf1-shRNA,检测了在腺病毒和shRNA介导下肝星状细胞系LX-2和HSC-T6中Sulf-1基因高表达和沉默时肝纤维化指标的变化,证明了Sulf-1基因能有效抑制肝星状细胞的激活和胶原蛋白的分泌。同时,我们进一步构建了人Sulf-1基因的N端活性片段的质粒载体pDC315-NSulf1,验证了NSulf-1与hSulf-1全长基因有类似的抗纤维化作用,为探索理想的抗纤维化基因治疗手段打下基础。 【研究方法】 1、分离培养并且鉴定原代肝星状细胞,检测原代肝星状细胞激活过程中Sulf-1基因表达水平的变化。 (1)采用肝脏原位灌注和密度梯度离心法分离Wistar大鼠肝脏的原代肝星状细胞。计数每只大鼠分离的细胞产量,台盼蓝检测分离的原代肝星状细胞的活力。 (2) Western Blot检测原代肝星状细胞中α-SMA、Desmin、GFAP、F4/80的表达,,达到鉴定原代肝星状细胞的目的。 (3)在原代肝星状细胞培养的0、3、5、7天,PCR检测原代肝星状细胞中Sulf-1基因的表达水平。 2、 MTT法检测重组TGF-β1因子对肝星状细胞系LX-2和HSC-T6增殖能力的影响, 检测重组TGF-β1因子刺激肝星状细胞系LX-2增殖的最适刺激浓度和刺激时间。 (1)检测不同时间跨度的重组TGF-β1因子对肝星状细胞系LX-2和HSC-T6增殖能力的影响。 (2)检测不同浓度梯度的重组TGF-β1因子对肝星状细胞系LX-2和HSC-T6增殖能力的影响。 3、构建hSulf-1基因的腺病毒载体Ad5-hSulf1及其N端活性片段腺质粒载体pDC315-NSulf1,构建沉默hSulf-1基因的shRNA载体hSulf1-shRNA,分别采用感染和转染的方法在肝星状细胞系LX-2中建立hSulf-1基因高表达和低表达的细胞系。 (1)不同病毒滴度的hSulf-1基因的腺病毒载体Ad5-hSulf1感染肝星状细胞系LX-2,RT-PCR检测不同病毒感染滴度下hSulf-1基因的表达,确定使hSulf-1基因高表达的最佳病毒感染滴度。 (2) hSulf1-shRNA转染肝星状细胞系LX-2,RT-PCR检测hSulf-1基因的沉默效果。 4、检测在重组TGF-β1因子刺激下的肝星状细胞系LX-2和HSC-T6中hSulf-1基因高表达及低表达时肝纤维化相关的效应指标的变化,验证NSulf-1基因对TGF-β1因子刺激下肝星状细胞系LX-2中肝纤维化相关的效应指标的变化的影响。 (1)重组TGF-β1因子刺激条件下,Ad5-Sulf1和hSulf1-shRNA干预肝星状细胞系LX-2和HSC-T6,PCR检测肝纤维化相关的效应指标α-SMA、CollagenⅠ、CollagenⅢ、TIMP-1的变化。 (2)重组TGF-β1因子刺激条件下,Ad5-Sulf1和pDC315-NSulf1干预肝星状细胞系LX-2和HSC-T6,PCR检测肝纤维化相关的效应指标α-SMA、CollagenⅠ、CollagenⅢ、TIMP-1的变化,Western-blot检测PCR检测肝纤维化相关的效应指标α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ的变化,检测信号通路分子p-ERK、p-AKT、p-Smad2的变化。 【实验结果】 1、 Western blot鉴定指标显示,正确分离出原代肝星状细胞。平均每只大鼠的原代肝星状细胞的产量为(3.008±0.3)×107个/只,细胞活力的平均值为(95.80±1.9)%。PCR结果显示,原代肝星状细胞在培养自发激活的过程中Sulf-1基因的表达水平逐渐下降。 2、刺激肝星状细胞系LX-2增殖的最佳重组TGF-β1因子干预条件为:5ng/ml,24h;刺激肝星状细胞系HSC-T6增殖的最佳重组TGF-β1因子干预条件为:7.5ng/ml,24h。 3、 Ad5-hSulf1感染肝星状细胞系LX-2的最佳病毒滴度为MOI=50,hSulf1-shRNA可以使肝星状细胞系LX-2中的Sulf1基因沉默,最佳转染条件为hSulf1-shRNA为2.5ug,Lipo2000Agent为7.5ul。 4、 Ad5-Sulf1能减弱重组TGF-β1因子刺激条件下,肝星状细胞系LX-2和HSC-T6中检测肝纤维化相关的效应指标α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ、TIMP-1的的表达,能减弱信号通路分子p-ERK、p-AKT、p-Smad2的表达。沉默hSulf-1基因对肝星状细胞系中肝纤维化相关的效应指标的作用不明显。pDC315-NSulf1具有与Ad5-hSulf1类似的抗纤维化作用。 【实验结论】 本实验分离鉴定了大鼠原代肝星状细胞,证明了Sulf-1基因的表达水平在原代肝星状细胞自发激活的过程中逐渐下降。构建了hSulf-1基因的腺病毒载体Ad5-hSulf1及其N端活性片段质粒载体pDC315-NSulf1,构建沉默hSulf-1基因的shRNA载体hSulf1-shRNA。检测了在重组TGF-β1因子刺激条件下,肝星状细胞系LX-2和HSC-T6中hSulf-1基因高表达及低表达时肝纤维化相关的效应指标的变化,证明hSulf-1基因在肝星状细胞系LX-2和HSC-T6中高表达时,能有效减弱肝纤维化相关的效应指标α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ、TIMP-1的的表达,能减弱信号通路分子p-ERK、p-AKT、p-Smad2的表达。证明了pDC315-NSulf1具有与Ad5-hSulf1类似的抗纤维化作用,为探索理想的抗纤维化基因治疗手段打下了基础。
[Abstract]:[research background and research purpose]
The pathophysiological process of liver fibrosis is closely related to various inflammatory mediators and cytokines in the liver microenvironment. The most important is the growth factors that promote the development of fibrosis. These growth factors mainly include TGF- beta 1, PDGF, CTGF, FGF, VEGF and so on. These growth factors play a role in both the cell surface and the cell surface. The growth factor receptor is binding and then activates the intracellular signaling pathway that is coupled with the receptor. When these growth factors are combined with the growth factor receptors on the cell surface, most of them also need to be a common ligand or provide a spatial conformation in the cell surface of the heparan sulfate proteoglycan (HSPG) molecule (HS). Quantitative studies have shown that the sulfation enzyme encoded by the Sulf-1 gene happens to desulfonate heparan sulfate in heparan sulfate proteoglycan molecules, thereby causing the heparin sulfate proteoglycan molecules to lose the ability to provide common ligands to growth factor receptors and to provide spatial image, thus blocking the growth factor signal. We conjecture that the Sulf-1 gene also blocks the signal transmission of growth factors related to the development and development of liver fibrosis, thus blocking the process of liver fibrosis. The expression level of Sulf-1 gene in rat primary hepatic stellate cells was detected with the change of the activation state of the primary hepatic stellate cells. The adenovirus vector Ad5-hSulf1 of human Sulf-1 gene was constructed, and the silent carrier of human Sulf-1 gene, hSulf1-shRNA, was used to detect the Sulf-1 gene in the hepatic stellate cell line LX-2 and HSC-T6 mediated by adenovirus and shRNA. The changes in the index of liver fibrosis in high expression and silence showed that the Sulf-1 gene could effectively inhibit the activation of hepatic stellate cells and the secretion of collagen. At the same time, we further constructed the plasmid vector pDC315-NSulf1 of the N terminal active fragment of human Sulf-1 gene, which proved that NSulf-1 and hSulf-1 full length genes have similar anti fibrosis effects. It lays the foundation for exploring the ideal anti fibrotic gene therapy.
[research methods]
1, isolation, culture and identification of primary hepatic stellate cells, and detect the expression level of Sulf-1 gene during activation of primary hepatic stellate cells.
(1) primary hepatic stellate cells were isolated from the liver of Wistar rats by liver perfusion and density gradient centrifugation. The cell output of each rat was counted, and trypan blue was used to detect the vitality of the isolated primary hepatic stellate cells.
(2) Western Blot detects the expression of alpha -SMA, Desmin, GFAP and F4/80 in primary hepatic stellate cells, and achieves the purpose of identifying primary hepatic stellate cells.
(3) the expression level of Sulf-1 gene in primary hepatic stellate cells was detected by PCR on the 0,3,5,7 day of primary hepatic stellate cell culture.
2, the effect of recombinant TGF- beta 1 factor on proliferation of hepatic stellate cell line LX-2 and HSC-T6 was detected by MTT.
The recombinant TGF- beta 1 was used to stimulate the proliferation and stimulation time of hepatic stellate cell line LX-2.
(1) to detect the effects of recombinant TGF- beta 1 at different time span on the proliferation of hepatic stellate cell line LX-2 and HSC-T6.
(2) to detect the effect of recombinant TGF- beta 1 with different concentration gradient on the proliferation of hepatic stellate cell line LX-2 and HSC-T6.
3, the adenovirus vector Ad5-hSulf1 of the hSulf-1 gene and its N terminal active fragment pDC315-NSulf1 were constructed to construct the shRNA carrier hSulf1-shRNA of the silent hSulf-1 gene, and the high expression and low expression of the hSulf-1 gene was established in the hepatic stellate cell line LX-2 by infection and transfection.
(1) the adenovirus vector Ad5-hSulf1 of the hSulf-1 gene of different virus titers infects the hepatic stellate cell line LX-2, and RT-PCR is used to detect the expression of hSulf-1 gene under the titer of different virus infection, and to determine the best virus infection titer that makes the hSulf-1 gene high expression.
(2) hSulf1-shRNA transfected hepatic stellate cell line LX-2 and RT-PCR to detect the silencing effect of hSulf-1 gene.
4, the changes in the effect of the high expression of hSulf-1 gene and low expression of liver fibrosis in the hepatic stellate cell lines LX-2 and HSC-T6 stimulated by recombinant TGF- beta factor were detected, and the effect of the NSulf-1 gene on the changes of hepatic fibrosis related index in the hepatic stellate cell line LX-2 under the stimulus of TGF- beta 1 was tested.
(1) under the conditions of recombinant TGF- beta 1 factor, Ad5-Sulf1 and hSulf1-shRNA interfered with the hepatic stellate cell line LX-2 and HSC-T6, and PCR was used to detect the effects of liver fibrosis, the changes of alpha -SMA, Collagen I, Collagen III and TIMP-1.
(2) under the conditions of recombinant TGF- beta 1 factor, Ad5-Sulf1 and pDC315-NSulf1 intervention in hepatic stellate cell line LX-2 and HSC-T6, PCR for the detection of liver fibrosis, the changes of alpha -SMA, Collagen I, Collagen III, TIMP-1, Western-blot detection of the effect of PCR in the detection of liver fibrosis. The changes of signal pathway molecules p-ERK, p-AKT and p-Smad2 were detected.
[experimental results]
1, the Western blot identification index showed that the primary hepatic stellate cells were correctly separated. The average output of primary hepatic stellate cells in each rat was (3.008 + 0.3) x 107 / only, and the mean value of cell viability was (95.80 + 1.9)%.PCR. The expression level of the primary hepatic stellate cells in the process of spontaneous activation of the primary hepatic stellate cells gradually decreased. Drop.
2, the best recombinant TGF- beta 1 factor intervention conditions for stimulating the proliferation of hepatic stellate cell line LX-2 are 5ng/ml, 24h; the best recombinant TGF- beta 1 factor for stimulating the proliferation of HSC-T6 in the hepatic stellate cell line is: 7.5ng/ml, 24h.
3, the optimal viral titer of Ad5-hSulf1 infected hepatic stellate cell line LX-2 is MOI=50. HSulf1-shRNA can silence the Sulf1 gene in the LX-2 of the hepatic stellate cell line. The optimal transfection condition is hSulf1-shRNA 2.5ug, Lipo2000Agent is 7.5ul..
4, Ad5-Sulf1 can weaken the expression of the effects of the liver fibrosis in the hepatic stellate cell line LX-2 and HSC-T6 under the stimulus of recombinant TGF- beta factor, the expression of alpha -SMA, TGF- beta 1, Collagen I, Collagen III, TIMP-1, which can weaken the expression of p-ERK, p-AKT, and p-Smad2. The effect of related indicators is not obvious..pDC315-NSulf1 has a similar anti fibrosis effect to Ad5-hSulf1.
[experimental conclusions]
In this experiment, the rat primary hepatic stellate cells were isolated and identified. It was proved that the expression level of Sulf-1 gene decreased gradually during the spontaneous activation of primary hepatic stellate cells. The adenovirus vector Ad5-hSulf1 of the hSulf-1 gene and its N terminal active fragment plasmid pDC315-NSulf1 were constructed, and the shRNA carrier hSulf1-shRNA. of the silent hSulf-1 gene was constructed. The effects of high expression of hSulf-1 gene and low expression of liver fibrosis in hepatic stellate cell line LX-2 and HSC-T6 were detected under the condition of recombinant TGF- beta 1 factor, which showed that the high expression of hSulf-1 gene in the hepatic stellate cell line LX-2 and HSC-T6 could effectively weaken the effect index of liver fibrosis, alpha -SMA, TGF- beta 1, Collag. The expression of en I, Collagen III, and TIMP-1 can weaken the expression of p-ERK, p-AKT and p-Smad2 in the signal pathway molecules. It is proved that pDC315-NSulf1 has the anti fibrosis effect similar to Ad5-hSulf1, which lays the foundation for exploring the ideal method of anti fibrosis gene therapy.
【学位授予单位】:第二军医大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R575.2

【参考文献】

相关期刊论文 前5条

1 HOU YunDe;;The hepatitis B vaccine originally jointly developed by China and the US is safe and effective[J];Science China(Life Sciences);2014年05期

2 Gunter Maubach;Michelle Chin Chia Lim;;GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line[J];World Journal of Gastroenterology;2006年05期

3 George Kolios;Vassilis Valatas;Elias Kouroumalis;;Role of Kupffer cells in the pathogenesis of liver disease[J];World Journal of Gastroenterology;2006年46期

4 Noriyuki Akutsu;Hiroyuki Yamamoto;Shigeru Sasaki;Hiroaki Taniguchi;Yoshiaki Arimura;Kohzoh Imai;Yasuhisa Shinomura;;Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma[J];World Journal of Gastroenterology;2010年28期

5 ;Notch3 regulates the activation of hepatic stellate cells[J];World Journal of Gastroenterology;2012年12期



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