發(fā)布時(shí)間：2018-06-08 06:33

  本文選題：肝纖維化 + 原代肝星狀細(xì)胞　； 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：【研究背景與研究目的】 肝纖維化的病理生理發(fā)展過(guò)程與肝臟微環(huán)境中的多種炎性介質(zhì)和細(xì)胞因子密切相關(guān)，其中最主要的就是各種促進(jìn)纖維化發(fā)生發(fā)展的生長(zhǎng)因子，這些生長(zhǎng)因子主要包括TGF-β1、PDGF、CTGF、FGF、VEGF等等，這些生長(zhǎng)因子發(fā)揮作用都需要和細(xì)胞表面的生長(zhǎng)因子受體結(jié)合，進(jìn)而激活與受體耦合的細(xì)胞內(nèi)信號(hào)通路。這些生長(zhǎng)因子和細(xì)胞表面的生長(zhǎng)因子受體結(jié)合時(shí)，大多還需要存在于細(xì)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）作為共同配體或提供空間構(gòu)象。而大量研究表明，Sulf-1基因編碼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發(fā)生去硫酸化，進(jìn)而使硫酸乙酰肝素蛋白聚糖分子失去為生長(zhǎng)因子受體提供共同配體和提供空間構(gòu)象的能力，從而阻斷生長(zhǎng)因子信號(hào)的傳遞。我們猜測(cè)，Sulf-1基因同樣能阻斷與促進(jìn)肝纖維化發(fā)生發(fā)展有關(guān)的生長(zhǎng)因子的信號(hào)傳遞，從而達(dá)到阻斷肝纖維化進(jìn)程的目的。目前對(duì)肝纖維化的研究結(jié)果顯示，在肝纖維化進(jìn)程中起關(guān)鍵作用的就是肝星狀細(xì)胞。于是，我們首先分離并檢測(cè)了大鼠原代肝星狀細(xì)胞中大鼠Sulf-1基因的表達(dá)水平隨原代肝星狀細(xì)胞激活狀態(tài)的變化，構(gòu)建了人Sulf-1基因的腺病毒載體Ad5-hSulf1，人Sulf-1基因的沉默載體hSulf1-shRNA，檢測(cè)了在腺病毒和shRNA介導(dǎo)下肝星狀細(xì)胞系LX-2和HSC-T6中Sulf-1基因高表達(dá)和沉默時(shí)肝纖維化指標(biāo)的變化，證明了Sulf-1基因能有效抑制肝星狀細(xì)胞的激活和膠原蛋白的分泌。同時(shí)，我們進(jìn)一步構(gòu)建了人Sulf-1基因的N端活性片段的質(zhì)粒載體pDC315-NSulf1，驗(yàn)證了NSulf-1與hSulf-1全長(zhǎng)基因有類似的抗纖維化作用，為探索理想的抗纖維化基因治療手段打下基礎(chǔ)。 【研究方法】 1、分離培養(yǎng)并且鑒定原代肝星狀細(xì)胞，檢測(cè)原代肝星狀細(xì)胞激活過(guò)程中Sulf-1基因表達(dá)水平的變化。 (1)采用肝臟原位灌注和密度梯度離心法分離Wistar大鼠肝臟的原代肝星狀細(xì)胞。計(jì)數(shù)每只大鼠分離的細(xì)胞產(chǎn)量，臺(tái)盼藍(lán)檢測(cè)分離的原代肝星狀細(xì)胞的活力。 (2) Western Blot檢測(cè)原代肝星狀細(xì)胞中α-SMA、Desmin、GFAP、F4/80的表達(dá)，，達(dá)到鑒定原代肝星狀細(xì)胞的目的。 (3)在原代肝星狀細(xì)胞培養(yǎng)的0、3、5、7天，PCR檢測(cè)原代肝星狀細(xì)胞中Sulf-1基因的表達(dá)水平。 2、 MTT法檢測(cè)重組TGF-β1因子對(duì)肝星狀細(xì)胞系LX-2和HSC-T6增殖能力的影響， 檢測(cè)重組TGF-β1因子刺激肝星狀細(xì)胞系LX-2增殖的最適刺激濃度和刺激時(shí)間。 (1)檢測(cè)不同時(shí)間跨度的重組TGF-β1因子對(duì)肝星狀細(xì)胞系LX-2和HSC-T6增殖能力的影響。 (2)檢測(cè)不同濃度梯度的重組TGF-β1因子對(duì)肝星狀細(xì)胞系LX-2和HSC-T6增殖能力的影響。 3、構(gòu)建hSulf-1基因的腺病毒載體Ad5-hSulf1及其N端活性片段腺質(zhì)粒載體pDC315-NSulf1，構(gòu)建沉默hSulf-1基因的shRNA載體hSulf1-shRNA，分別采用感染和轉(zhuǎn)染的方法在肝星狀細(xì)胞系LX-2中建立hSulf-1基因高表達(dá)和低表達(dá)的細(xì)胞系。 (1)不同病毒滴度的hSulf-1基因的腺病毒載體Ad5-hSulf1感染肝星狀細(xì)胞系LX-2，RT-PCR檢測(cè)不同病毒感染滴度下hSulf-1基因的表達(dá)，確定使hSulf-1基因高表達(dá)的最佳病毒感染滴度。 (2) hSulf1-shRNA轉(zhuǎn)染肝星狀細(xì)胞系LX-2，RT-PCR檢測(cè)hSulf-1基因的沉默效果。 4、檢測(cè)在重組TGF-β1因子刺激下的肝星狀細(xì)胞系LX-2和HSC-T6中hSulf-1基因高表達(dá)及低表達(dá)時(shí)肝纖維化相關(guān)的效應(yīng)指標(biāo)的變化，驗(yàn)證NSulf-1基因?qū)GF-β1因子刺激下肝星狀細(xì)胞系LX-2中肝纖維化相關(guān)的效應(yīng)指標(biāo)的變化的影響。 (1)重組TGF-β1因子刺激條件下，Ad5-Sulf1和hSulf1-shRNA干預(yù)肝星狀細(xì)胞系LX-2和HSC-T6，PCR檢測(cè)肝纖維化相關(guān)的效應(yīng)指標(biāo)α-SMA、CollagenⅠ、CollagenⅢ、TIMP-1的變化。 (2)重組TGF-β1因子刺激條件下，Ad5-Sulf1和pDC315-NSulf1干預(yù)肝星狀細(xì)胞系LX-2和HSC-T6，PCR檢測(cè)肝纖維化相關(guān)的效應(yīng)指標(biāo)α-SMA、CollagenⅠ、CollagenⅢ、TIMP-1的變化，Western-blot檢測(cè)PCR檢測(cè)肝纖維化相關(guān)的效應(yīng)指標(biāo)α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ的變化，檢測(cè)信號(hào)通路分子p-ERK、p-AKT、p-Smad2的變化。 【實(shí)驗(yàn)結(jié)果】 1、 Western blot鑒定指標(biāo)顯示，正確分離出原代肝星狀細(xì)胞。平均每只大鼠的原代肝星狀細(xì)胞的產(chǎn)量為(3.008±0.3)×107個(gè)/只，細(xì)胞活力的平均值為(95.80±1.9)%。PCR結(jié)果顯示，原代肝星狀細(xì)胞在培養(yǎng)自發(fā)激活的過(guò)程中Sulf-1基因的表達(dá)水平逐漸下降。 2、刺激肝星狀細(xì)胞系LX-2增殖的最佳重組TGF-β1因子干預(yù)條件為：5ng/ml，24h；刺激肝星狀細(xì)胞系HSC-T6增殖的最佳重組TGF-β1因子干預(yù)條件為：7.5ng/ml，24h。 3、 Ad5-hSulf1感染肝星狀細(xì)胞系LX-2的最佳病毒滴度為MOI=50，hSulf1-shRNA可以使肝星狀細(xì)胞系LX-2中的Sulf1基因沉默，最佳轉(zhuǎn)染條件為hSulf1-shRNA為2.5ug，Lipo2000Agent為7.5ul。 4、 Ad5-Sulf1能減弱重組TGF-β1因子刺激條件下，肝星狀細(xì)胞系LX-2和HSC-T6中檢測(cè)肝纖維化相關(guān)的效應(yīng)指標(biāo)α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ、TIMP-1的的表達(dá)，能減弱信號(hào)通路分子p-ERK、p-AKT、p-Smad2的表達(dá)。沉默hSulf-1基因?qū)Ω涡菭罴?xì)胞系中肝纖維化相關(guān)的效應(yīng)指標(biāo)的作用不明顯。pDC315-NSulf1具有與Ad5-hSulf1類似的抗纖維化作用。 【實(shí)驗(yàn)結(jié)論】 本實(shí)驗(yàn)分離鑒定了大鼠原代肝星狀細(xì)胞，證明了Sulf-1基因的表達(dá)水平在原代肝星狀細(xì)胞自發(fā)激活的過(guò)程中逐漸下降。構(gòu)建了hSulf-1基因的腺病毒載體Ad5-hSulf1及其N端活性片段質(zhì)粒載體pDC315-NSulf1，構(gòu)建沉默hSulf-1基因的shRNA載體hSulf1-shRNA。檢測(cè)了在重組TGF-β1因子刺激條件下，肝星狀細(xì)胞系LX-2和HSC-T6中hSulf-1基因高表達(dá)及低表達(dá)時(shí)肝纖維化相關(guān)的效應(yīng)指標(biāo)的變化，證明hSulf-1基因在肝星狀細(xì)胞系LX-2和HSC-T6中高表達(dá)時(shí)，能有效減弱肝纖維化相關(guān)的效應(yīng)指標(biāo)α-SMA、TGF-β1、CollagenⅠ、CollagenⅢ、TIMP-1的的表達(dá)，能減弱信號(hào)通路分子p-ERK、p-AKT、p-Smad2的表達(dá)。證明了pDC315-NSulf1具有與Ad5-hSulf1類似的抗纖維化作用，為探索理想的抗纖維化基因治療手段打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.2

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本文編號(hào)：1994952