本文選題：肝纖維化 + 肝星狀細(xì)胞��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：肝纖維化（hepatic fibrosis）是多種細(xì)胞及介質(zhì)參與的，針對(duì)各種肝損傷的損傷修復(fù)反應(yīng)。肝纖維化的主要特征是以Ⅰ型膠原蛋白為主的細(xì)胞外基質(zhì)（extracellular matrix, ECM）成分異常沉積。Ⅰ型膠原蛋白主要來源于活化的肝星狀細(xì)胞（hepatic stellate cell，HSC）。Ⅰ型膠原蛋白是由兩條α1鏈和一條α2鏈構(gòu)成的異三聚體，呈三螺旋結(jié)構(gòu)，COL1A1和COL1A2基因分別編碼其α1鏈和α2鏈。LX-2細(xì)胞是人的肝星狀細(xì)胞系。 一、HBV對(duì)肝星狀細(xì)胞Ⅰ型膠原蛋白表達(dá)調(diào)控研究 HBV感染者可以發(fā)生肝纖維化，但其分子機(jī)制仍未完全闡明。許多研究表明HBV感染肝細(xì)胞后，肝細(xì)胞分泌轉(zhuǎn)化生長因子β1（transforming growthfactor-β1，TGFβ1）等細(xì)胞因子，這些細(xì)胞因子作用于HSC膜受體，引起HSC活化。但涉及到HBV對(duì)HSC直接作用的研究并不多，很多問題有待解決，諸如HBV能否感染HSC，HBV是否影響HSCⅠ型膠原蛋白的表達(dá)，其機(jī)制如何。本研究第一部分即針對(duì)這些問題進(jìn)行研究。 1. HBV對(duì)LX-2細(xì)胞的感染及Ⅰ型膠原蛋白表達(dá)的影響 HBV能否感染LX-2細(xì)胞。將LX-2細(xì)胞與HBV病毒共培養(yǎng)，24h后更換正常培養(yǎng)液。在其后0h、12h、24h分別收集細(xì)胞并裂解，ELISA檢測(cè)細(xì)胞內(nèi)HBs抗原、HBe抗原。在其后24h，免疫組化檢測(cè)細(xì)胞內(nèi)HBc抗原，，HBV DNA定量檢測(cè)細(xì)胞內(nèi)HBV DNA。結(jié)果：在HBV共培養(yǎng)LX-2細(xì)胞中檢測(cè)到HBs抗原、HBe抗原、HBc抗原的表達(dá)及HBV DNA的存在。這些結(jié)果說明HBV可以感染進(jìn)入LX-2細(xì)胞。 HBV對(duì)Ⅰ型膠原蛋白表達(dá)的影響。肝纖維化的主要特征是以Ⅰ型膠原蛋白為主的ECM成分過度沉積，本部分以Ⅰ型膠原α1鏈為靶標(biāo)研究HBV致肝纖維化作用。將HBV真核表達(dá)載體和空載體分別轉(zhuǎn)染LX-2細(xì)胞，48h后收集細(xì)胞，Western Blot檢測(cè)Ⅰ型膠原蛋白表達(dá)。結(jié)果：HBV表達(dá)組Ⅰ型膠原蛋白表達(dá)明顯高于對(duì)照組。這說明在LX-2細(xì)胞中，HBV可以促進(jìn)Ⅰ型膠原蛋白表達(dá)。 2. HBV編碼蛋白對(duì)LX-2細(xì)胞Ⅰ型膠原蛋白表達(dá)的影響 HBV編碼蛋白對(duì)COL1A1表達(dá)的影響。上述研究中HBV可以促進(jìn)Ⅰ型膠原蛋白α1鏈表達(dá)，為了探索其分子機(jī)制，首先要確定HBV促進(jìn)Ⅰ型膠原α1鏈表達(dá)的病毒成分。將HBV各編碼蛋白真核表達(dá)載體和空載體分別轉(zhuǎn)染LX-2細(xì)胞，48h后收集細(xì)胞。real-time PCR定量檢測(cè)COL1A1mRNA相對(duì)表達(dá)，Western Blot檢測(cè)Ⅰ型膠原蛋白表達(dá)。結(jié)果表明HBV各編碼蛋白不同程度地促進(jìn)COL1A1mRNA和蛋白表達(dá)，其中HBx的作用最強(qiáng)。 HBV編碼蛋白對(duì)COL1A1啟動(dòng)子活性的影響。前述HBV各編碼蛋白均可從轉(zhuǎn)錄水平促進(jìn)COL1A1表達(dá)，推測(cè)其可能通過上調(diào)COL1A1啟動(dòng)子活性促進(jìn)COL1A1轉(zhuǎn)錄表達(dá)。將HBV各編碼蛋白真核表達(dá)載體和空載體分別與COL1A1啟動(dòng)子報(bào)告基因載體及內(nèi)參報(bào)告基因載體共轉(zhuǎn)染LX-2細(xì)胞，24h后收集細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子活性。結(jié)果證實(shí)HBV各編碼蛋白不同程度地上調(diào)COL1A1啟動(dòng)子活性，其中HBx的作用最強(qiáng)。 3. HBx對(duì)LX-2細(xì)胞Ⅰ型膠原蛋白表達(dá)的調(diào)控機(jī)制研究 HBV感染LX-2細(xì)胞后HBx在細(xì)胞中的表達(dá)。上述結(jié)果發(fā)現(xiàn)HBx蛋白可以上調(diào)LX-2細(xì)胞COL1A1啟動(dòng)子活性進(jìn)而促進(jìn)COL1A1表達(dá)，且作用強(qiáng)于其它的HBV編碼蛋白，以往研究尚無相關(guān)結(jié)果的報(bào)道，因此選擇HBx進(jìn)行深入研究。在研究HBx調(diào)控Ⅰ型膠原蛋白表達(dá)的機(jī)制之前，需要明確HBV感染LX-2細(xì)胞后HBx能否在細(xì)胞中表達(dá)。將LX-2細(xì)胞與不同濃度的HBV病毒共培養(yǎng)，48h后去除培養(yǎng)上清,收集并洗滌細(xì)胞后裂解，RT-PCR半定量檢測(cè)HBx mRNA表達(dá)，Western Blot檢測(cè)HBx蛋白表達(dá)。結(jié)果：隨著共培養(yǎng)的HBV濃度升高，HBx mRNA及蛋白表達(dá)增加。這說明HBV感染LX-2細(xì)胞后HBx蛋白可以在細(xì)胞中表達(dá)，且其表達(dá)量與HBV濃度正相關(guān)。 COL1A1啟動(dòng)子上受HBx調(diào)控的活性區(qū)域。將COL1A1啟動(dòng)子全長分成兩段：F1段（-2483～-269bp）和F2段（-268～+42bp），分別構(gòu)建報(bào)告基因載體。將構(gòu)建的含COL1A1啟動(dòng)子各段及全長報(bào)告基因載體分別與內(nèi)參報(bào)告基因載體組合共轉(zhuǎn)染LX-2細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子各段及全長活性。結(jié)果：F2段活性大于F1段活性。將HBx真核表達(dá)載體和空載體分別與COL1A1啟動(dòng)子F2段及全長報(bào)告基因載體組合轉(zhuǎn)染LX-2細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子F2段及全長活性。結(jié)果：HBx除了促進(jìn)全長啟動(dòng)子活性，也可以促進(jìn)COL1A1啟動(dòng)子F2段活性。以上結(jié)果說明： COL1A1啟動(dòng)子F2段（-268～+42bp）是受HBx調(diào)控的活性區(qū)域，這為進(jìn)一步研究其調(diào)控通路提供了依據(jù)。 HBx調(diào)控COL1A1表達(dá)過程中TGFβ1的作用。TGFβ1在HSC的活化及COL1A1表達(dá)過程中發(fā)揮重要作用。為了研究TGFβ1是否在HBx調(diào)控COL1A1表達(dá)過程中起作用，使用定量PCR檢測(cè)HBx是否促進(jìn)TGFβ1的表達(dá)，雙報(bào)告基因法檢測(cè)抑制TGFβ1的表達(dá)是否影響HBx對(duì)COL1A1啟動(dòng)子活性的上調(diào)作用。結(jié)果：HBx不促進(jìn)TGFβ1的表達(dá)；抑制TGFβ1的表達(dá)不影響HBx對(duì)COL1A1啟動(dòng)子活性的上調(diào)作用。這說明TGFβ1不參與HBx對(duì)Ⅰ型膠原蛋白表達(dá)的調(diào)控。賈繼東等研究結(jié)果：用TGFβ1中和抗體阻斷TGFβ1與受體的結(jié)合不影響HBV對(duì)LX-2細(xì)胞Ⅰ型膠原蛋白表達(dá)的促進(jìn)作用。這與本研究的結(jié)果一致。 HBx調(diào)控COL1A1表達(dá)過程中P38MAPK通路的作用。P38MAPK通路在HBV誘導(dǎo)的COL1A1表達(dá)過程中發(fā)揮作用。為了研究HBx對(duì)COL1A1啟動(dòng)子的上調(diào)作用是否通過P38MAPK通路實(shí)現(xiàn)，將HBx真核表達(dá)載體和空載體分別與COL1A1啟動(dòng)子報(bào)告基因載體及內(nèi)參報(bào)告基因載體共轉(zhuǎn)染LX-2細(xì)胞，6h后，加P38MAPK通路阻斷劑SB203580，24h后收集細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子活性。結(jié)果：阻斷P38MAPK通路后，HBx對(duì)COL1A1啟動(dòng)子活性的上調(diào)作用被抑制。這說明P38MAPK通路參與HBx對(duì)Ⅰ型膠原蛋白表達(dá)的調(diào)控。 二、新型抗纖維化藥物篩選模型研究 肝纖維化的主要特征是以Ⅰ型膠原蛋白為主的ECM成分過度沉積，故抑制Ⅰ型膠原表達(dá)是目前抗肝纖維化治療的重要策略之一。轉(zhuǎn)錄水平基因啟動(dòng)子活性的調(diào)節(jié)是基因表達(dá)調(diào)控的關(guān)鍵環(huán)節(jié)之一，本課題第一部分的研究結(jié)果也能很好的說明這一點(diǎn)，即COL1A1mRNA及蛋白表達(dá)與COL1A1啟動(dòng)子活性具有很好的對(duì)應(yīng)關(guān)系。本研究第二部分?jǐn)M以COL1A1啟動(dòng)子報(bào)告基因系統(tǒng)為基礎(chǔ)建立抑制Ⅰ型膠原蛋白表達(dá)的抗纖維化藥物篩選和評(píng)價(jià)模型。 1.抗纖維化藥物篩選模型的建立 COL1A1啟動(dòng)子報(bào)告基因載體的構(gòu)建。根據(jù)COL1A1基因上游的序列設(shè)計(jì)合成擴(kuò)增引物，以人基因組DNA為模板，PCR擴(kuò)增COL1A1啟動(dòng)子序列，將其插入報(bào)告基因載體pGL4中，構(gòu)建COL1A1啟動(dòng)子報(bào)告基因載體pGL4-COL1A1promoter，限制性酶切和DNA測(cè)序鑒定其正確性。 抗纖維化藥物篩選模型的建立及檢測(cè)。以LX-2細(xì)胞為宿主細(xì)胞，將COL1A1啟動(dòng)子報(bào)告基因載體與內(nèi)參報(bào)告基因載體共轉(zhuǎn)染LX-2細(xì)胞，24h后收集細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子活性�？估w維化藥物篩選模型建立之后，使用對(duì)Ⅰ型膠原蛋白表達(dá)有抑制作用的TGFβ1siRNA驗(yàn)證其有效性。結(jié)果：TGFβ1siRNA不影響COL1A1啟動(dòng)子活性。分析其原因：一方面可能與細(xì)胞內(nèi)源性TGFβ1表達(dá)低下有關(guān)；另一方面與正常LX-2細(xì)胞COL1A1啟動(dòng)子處于低活化狀態(tài)有關(guān)。擬外源表達(dá)活化劑TGFβ1活化LX-2細(xì)胞，模擬肝纖維化肝星狀細(xì)胞的高活化狀態(tài)，再進(jìn)行藥物評(píng)價(jià)實(shí)驗(yàn)。 2.抗纖維化藥物篩選模型的優(yōu)化 抗纖維化藥物篩選模型的首次優(yōu)化。以HepG2細(xì)胞提取的總RNA逆轉(zhuǎn)錄產(chǎn)物為模板，PCR擴(kuò)增TGFβ1全長編碼基因，插入到表達(dá)載體pcDNA3.1中，構(gòu)建TGFβ1的重組載體pcDNA3.1-TGFβ1，限制性酶切和DNA測(cè)序鑒定為正確連接的克隆。將pcDNA3.1、pcDNA3.1-TGFβ1分別與COL1A1啟動(dòng)子報(bào)告基因載體及內(nèi)參報(bào)告基因載體共轉(zhuǎn)染LX-2細(xì)胞，24h后收集細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子活性。結(jié)果：過表達(dá)TGFβ1僅使COL1A1啟動(dòng)子活性提高了不到2倍（t=3.396，P=0.0274）。推測(cè)其原因：可能是TGFβ1在細(xì)胞內(nèi)表達(dá)但沒有分泌到細(xì)胞外，無法與細(xì)胞膜受體結(jié)合進(jìn)而發(fā)揮作用。擬構(gòu)建含分泌信號(hào)肽的TGFβ1重組表達(dá)載體，驗(yàn)證上述推測(cè)是否正確。 抗纖維化藥物篩選模型的再次優(yōu)化。同上方法構(gòu)建含分泌信號(hào)肽的TGFβ1重組表達(dá)載體pJW4303-TGFβ1。將pJW4303、pJW4303-TGFβ1分別與COL1A1啟動(dòng)子報(bào)告基因載體及內(nèi)參報(bào)告基因載體共轉(zhuǎn)染LX-2細(xì)胞，24h后收集細(xì)胞，雙報(bào)告基因檢測(cè)COL1A1啟動(dòng)子活性。結(jié)果：分泌表達(dá)TGFβ1使COL1A1啟動(dòng)子活性提高了200倍以上（t=21.78，P=0.0001）。該結(jié)果驗(yàn)證了上述推測(cè)：TGFβ1只有被分泌到細(xì)胞外并與自身細(xì)胞膜受體結(jié)合后才能發(fā)揮活化作用。故選擇pJW4303-TGFβ1用于抗纖維化藥物篩選模型的優(yōu)化。 3.抗纖維化藥物篩選模型的有效性鑒定 文獻(xiàn)報(bào)道糖皮質(zhì)激素類藥物地塞米松可以下調(diào)COL1A1啟動(dòng)子活性進(jìn)而抑制Ⅰ型膠原蛋白表達(dá)，故選其作為陽性模式藥物來鑒定該篩選模型的有效性。在高活化狀態(tài)的篩選模型中，加入不同濃度地塞米松，觀察其抑制效果。結(jié)果：地塞米松對(duì)COL1A1啟動(dòng)子活性具有明顯的抑制作用且具有劑量依賴關(guān)系。這說明該模型可以用于篩選和評(píng)價(jià)抑制I型膠原蛋白表達(dá)的抗纖維化藥物。 本研究證實(shí)了HBV能感染LX-2細(xì)胞并促進(jìn)Ⅰ型膠原蛋白表達(dá)；HBV編碼蛋白尤其是HBx能上調(diào)COL1A1啟動(dòng)子活性進(jìn)而促進(jìn)Ⅰ型膠原蛋白表達(dá)；TGFβ1不參與而P38MAPK通路參與HBx對(duì)Ⅰ型膠原蛋白的調(diào)控。另外，本研究以COL1A1啟動(dòng)子活性調(diào)控機(jī)制為基礎(chǔ)，建立了一種新型抗纖維化藥物篩選模型，為抑制COL1A1啟動(dòng)子活性進(jìn)而抑制Ⅰ型膠原蛋白表達(dá)的抗纖維化藥物研究奠定了基礎(chǔ)。
[Abstract]:Hepatic fibrosis is involved in a variety of cells and mediators to repair the damage of various liver injuries. The main feature of liver fibrosis is the abnormal deposition of extracellular matrix (ECM) based on type I collagen. Type I collagen egg white mainly comes from the activated hepatic stellate cells (hepatic stell). Ate cell, HSC). Type I collagen is an ISO trimer consisting of two alpha 1 chains and one 2 chain, which is a three helix structure. The COL1A1 and COL1A2 genes encode their alpha 1 chain and alpha 2 chain.LX-2 cells respectively.
1. HBV regulates the expression of type I collagen in hepatic stellate cells.
Liver fibrosis can occur in HBV infected people, but its molecular mechanism is still not fully elucidated. Many studies have shown that after HBV infection of liver cells, hepatocytes secrete cytokines such as transforming growth factor beta 1 (transforming growthfactor- beta 1, TGF beta 1). These cytokines play a role in HSC membrane receptors and cause HSC activation. But it involves the study of HBV's direct effect on HSC. There are not many problems, many problems need to be solved, such as whether HBV can infect HSC, whether HBV affects the expression of HSC type I collagen and its mechanism. The first part of this study is to study these problems.
Effect of 1. HBV on LX-2 cell infection and expression of type I collagen
HBV can infect LX-2 cells. Co culture LX-2 cells with HBV virus, and then replace normal culture medium after 24h. After 0h, 12h, 24h are collected and lysed respectively. ELISA detection of intracellular HBs antigen, HBe antigen. The presence of HBs antigen, HBe antigen, HBc antigen and HBV DNA were detected. These results indicate that HBV can infect LX-2 cells.
The effect of HBV on the expression of type I collagen. The main feature of liver fibrosis is the excessive deposition of ECM components based on type I collagen. This part studies the role of HBV induced liver fibrosis with the type I collagen alpha 1 chain as a target. The HBV eukaryotic expression vector and empty vector are transfected to LX-2 cells respectively, and the cells are collected after 48h and Western Blot is used to detect type I glue. Results: the expression of type I collagen in the HBV expression group was significantly higher than that in the control group. This indicates that HBV can promote the expression of type I collagen in LX-2 cells.
Effect of 2. HBV encoding protein on type I collagen expression in LX-2 cells
The effect of HBV encoding protein on COL1A1 expression. In the above study, HBV can promote the expression of type I collagen alpha 1 chain. In order to explore its molecular mechanism, first we must determine the virus component that HBV promotes the expression of type I collagen alpha 1 chain. HBV each encoded protein eukaryotic expression vector and empty vector are transfected to LX-2 cells respectively, and.Real-time PCR is collected after 48h. The relative expression of COL1A1mRNA was detected, and the expression of type I collagen was detected by Western Blot. The results showed that each encoding protein of HBV promoted the expression of COL1A1mRNA and protein in varying degrees, and the effect of HBx was the strongest.
The effect of HBV encoding protein on the activity of COL1A1 promoter. The previous HBV encoding proteins can promote the expression of COL1A1 from the transcriptional level, presumably by up-regulation the activity of COL1A1 promoter to promote COL1A1 transcriptional expression. The eukaryotic expression vector and the empty vector of the HBV encoded proteins and the COL1A1 promoter report gene carrier and the internal reference base, respectively. The LX-2 cells were co transfected with the carrier, and the cells were collected after 24h, and the double reporter gene was used to detect the activity of COL1A1 promoter. The results showed that the COL1A1 promoter activity was up regulated by each HBV protein in varying degrees, and HBx was the strongest.
Regulatory mechanism of 3. HBx on the expression of type I collagen in LX-2 cells
The expression of HBx in LX-2 cells after HBV infection. The results show that HBx protein can up regulate the activity of LX-2 cell COL1A1 promoter and promote the expression of COL1A1, and it is stronger than other HBV encoded proteins. There is no related results in previous studies. Therefore, HBx is selected for in-depth study. In the study of HBx regulation of type I collagen expression Before the mechanism of HBV infection, it is necessary to make clear whether HBx can be expressed in cells after infection of LX-2 cells. LX-2 cells are co cultured with HBV virus of different concentrations, 48h is used to remove culture supernatant, and then cleavage after cells are collected and washed, RT-PCR semi quantitative detection of HBx mRNA expression, Western Blot detection of HBx egg white expression. X mRNA and protein expression increased. This indicates that HBx can be expressed in cells after HBV infection with LX-2 cells, and its expression is positively correlated with HBV concentration.
The active region of the COL1A1 promoter was regulated by HBx. The total length of COL1A1 promoter was divided into two segments: F1 segment (-2483 to -269bp) and F2 segment (-268 to +42bp), and the reporter gene vector was constructed respectively. The construction of all the COL1A1 promoters and the full length reporter gene vector were co transfected to the LX-2 cell with the internal reference reporter gene vector, and the double reporter gene was transfected. The activity of all segments and full length of COL1A1 promoter was detected. Results: the activity of F2 segment was greater than that of F1 segment. HBx eukaryotic expression vector and empty carrier were transfected into LX-2 cells with COL1A1 promoter F2 segment and full length reporter gene vector respectively, and the F2 segment and full length activity of COL1A1 promoter were detected by double reporter gene. The results: HBx promoted the activity of the full-length promoter in addition to HBx. It can also promote the activity of the F2 segment of the COL1A1 promoter. The above results indicate that the F2 segment of the COL1A1 promoter (-268 to +42bp) is the active region regulated by HBx, which provides a basis for further study of its regulatory pathway.
HBx regulates the role of TGF beta 1 in the process of COL1A1 expression..TGF beta 1 plays an important role in the activation of HSC and the expression of COL1A1. In order to study whether TGF beta 1 plays a role in the regulation of COL1A1 expression in HBx, quantitative PCR is used to detect whether HBx promotes the expression of TGF beta 1. The up-regulation effect of promoter activity. Results: HBx does not promote the expression of TGF beta 1; inhibition of the expression of TGF beta 1 does not affect the up-regulation of HBx on COL1A1 promoter activity. This indicates that TGF beta 1 does not participate in the regulation of the expression of type I collagen by HBx. The results of Jia Jidong and other studies: the binding of TGF beta 1 neutralizing antibodies to the binding of TGF beta 1 to the receptor does not affect HBV The expression of type I collagen was promoted by LX-2 cells. This is consistent with the results of this study.
HBx regulates the role of P38MAPK pathway in the process of COL1A1 expression..P38MAPK pathway plays a role in HBV induced COL1A1 expression. In order to study whether the up regulation of HBx on COL1A1 promoter is realized through the P38MAPK pathway, HBx eukaryotic expression vector and empty carrier and COL1A1 promoter report gene carrier and gene report gene carrier, respectively. The body co transfected LX-2 cells, after 6h, adding P38MAPK pathway blocker SB203580,24h to collect cells and double reporter gene to detect COL1A1 promoter activity. Results: after blocking P38MAPK pathway, the up-regulation effect of HBx on COL1A1 promoter activity was inhibited. This shows that P38MAPK pathway participates in the regulation of the expression of collagen type I by HBx.
Two, study on a new screening model for anti fibrosis drugs
The main feature of liver fibrosis is the overdeposition of ECM components based on type I collagen. Therefore, inhibiting the expression of type I collagen is one of the important strategies for the treatment of liver fibrosis. The regulation of the promoter activity of the transcriptional gene is one of the key links in the regulation of gene expression. The results of the first part of this subject are also very good. The second part of this study is based on the establishment of the COL1A1 promoter reporting gene system to establish a screening and evaluation model for anti fibrosis drugs that inhibit the expression of type I collagen protein in the second part of this study.
The establishment of 1. anti fibrotic drug screening model
COL1A1 promoter reporter gene vector construction. Based on the sequence of COL1A1 gene upstream sequence design and amplification primers, the human genome DNA as the template, PCR amplification COL1A1 promoter sequence, and insert it into the report gene carrier pGL4, construct the COL1A1 promoter reporter gene carrier pGL4-COL1A1promoter, restriction enzyme cut and DNA sequencing to identify its correctness Sex.
The establishment and detection of the anti fibrosis drug screening model. Using LX-2 cells as host cells, the COL1A1 promoter reporter gene carrier and the internal reference reporter gene were co transfected to LX-2 cells, the cells were collected after 24h and the double reporter gene was used to detect the activity of COL1A1 promoter. After the establishment of the anti fibrosis drug screening model, the expression of type I collagen was used. The inhibitory effect of TGF beta 1siRNA was verified. Results: TGF beta 1siRNA did not affect the activity of COL1A1 promoter. The reason is that it may be related to the low expression of endogenous TGF beta 1; on the other hand, it is related to the low activation state of COL1A1 promoter in normal LX-2 cells. The activation agent TGF beta 1 activates LX-2 cells, The high activation state of hepatic stellate cells was simulated, and then the drug evaluation experiment was carried out.
Optimization of 2. anti fibrotic drug screening model
The first optimization of the anti fibrosis drug screening model. Using the total RNA reverse product extracted by HepG2 cells as a template, PCR amplified TGF beta 1 full length encoding gene, inserted into the expression vector pcDNA3.1, and constructed the recombinant vector of TGF beta 1, pcDNA3.1-TGF beta 1. Restrictive enzyme digestion and DNA sequencing were identified as the correct clones. PcDNA3.1, pcDNA3.1-TGF beta 1. Don't co transfect LX-2 cells with COL1A1 promoter reporter gene carrier and internal reference reporter gene carrier, collect cells after 24h and double reporter gene to detect the activity of COL1A1 promoter. Results: overexpression of TGF beta 1 only makes COL1A1 promoter activity less than 2 times (t=3.396, P=0.0274). It is presumed that the reason is that TGF beta 1 is expressed in cell but not in cell The expression of TGF beta 1 recombinant expression vector containing the secretory signal peptide was constructed to verify the correctness of the conjecture.
The anti fibrosis drug screening model was reoptimized. TGF beta 1 recombinant expression vector containing the secretory signal peptide was constructed with pJW4303-TGF beta 1., pJW4303, pJW4303-TGF beta 1 were co transfected to LX-2 cells with COL1A1 promoter reporter gene carrier and the internal reference reporter gene vector respectively. After 24h, the cells were collected, and the double reporter gene was used to detect the activity of COL1A1 promoter. Results: the secretory expression of TGF beta 1 increased the activity of COL1A1 promoter more than 200 times (t=21.78, P=0.0001). The results showed that TGF beta 1 was only secreted outside the cell and combined with its own cell membrane receptor to play an active role. Therefore, the selection of pJW4303-TGF beta 1 was optimized for the screening model of anti fibrosis drugs.
Effectiveness identification of 3. anti fibrosis drug screening model
It is reported that glucocorticoid dexamethasone can downregulate the activity of COL1A1 promoter and inhibit the expression of type I collagen. Therefore, it is selected as a positive model drug to identify the effectiveness of the screening model. In the screening model of high activation state, the inhibitory effect of different concentrations of sliamethasone is added to the results: dexamethasone. It has a significant inhibitory effect on the activity of COL1A1 promoter and has a dose-dependent manner, which indicates that the model can be used to screen and evaluate anti fibrosis drugs that inhibit the expression of I type collagen.
This study confirmed that HBV can infect LX-2 cells and promote the expression of type I collagen; HBV encoded protein, especially HBx, can up-regulate the activity of COL1A1 promoter and promote the expression of type I collagen; TGF beta 1 is not involved while P38MAPK pathway participates in the regulation of HBx to type I collagen. In addition, this study is based on the regulation mechanism of COL1A1 promoter activity. On the basis of this, a new screening model for anti fibrosis drugs was established, which laid the foundation for the study of anti fibrosis drugs that inhibit the activity of COL1A1 promoter and inhibit the expression of type I collagen.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.62;R575.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Hepatitis B virus X protein promotes proliferation and upregulates TGF-β1 and CTGF in human hepatic stellate cell line,LX-2[J];Hepatobiliary & Pancreatic Diseases International;2009年01期

本文編號(hào)：2053112