HBV对肝星状细胞Ⅰ型胶原蛋白表达调控及新型抗纤维化药物筛选模型研究

发布时间：2018-06-22 14:21

本文选题：肝纤维化 + 肝星状细胞　；参考：《中国人民解放军军事医学科学院》2014年硕士论文

【摘要】：肝纤维化（hepatic fibrosis）是多种细胞及介质参与的，针对各种肝损伤的损伤修复反应。肝纤维化的主要特征是以Ⅰ型胶原蛋白为主的细胞外基质（extracellular matrix, ECM）成分异常沉积。Ⅰ型胶原蛋白主要来源于活化的肝星状细胞（hepatic stellate cell，HSC）。Ⅰ型胶原蛋白是由两条α1链和一条α2链构成的异三聚体，呈三螺旋结构，COL1A1和COL1A2基因分别编码其α1链和α2链。LX-2细胞是人的肝星状细胞系。一、HBV对肝星状细胞Ⅰ型胶原蛋白表达调控研究 HBV感染者可以发生肝纤维化，但其分子机制仍未完全阐明。许多研究表明HBV感染肝细胞后，肝细胞分泌转化生长因子β1（transforming growthfactor-β1，TGFβ1）等细胞因子，这些细胞因子作用于HSC膜受体，引起HSC活化。但涉及到HBV对HSC直接作用的研究并不多，很多问题有待解决，诸如HBV能否感染HSC，HBV是否影响HSCⅠ型胶原蛋白的表达，其机制如何。本研究第一部分即针对这些问题进行研究。 1. HBV对LX-2细胞的感染及Ⅰ型胶原蛋白表达的影响 HBV能否感染LX-2细胞。将LX-2细胞与HBV病毒共培养，24h后更换正常培养液。在其后0h、12h、24h分别收集细胞并裂解，ELISA检测细胞内HBs抗原、HBe抗原。在其后24h，免疫组化检测细胞内HBc抗原，，HBV DNA定量检测细胞内HBV DNA。结果：在HBV共培养LX-2细胞中检测到HBs抗原、HBe抗原、HBc抗原的表达及HBV DNA的存在。这些结果说明HBV可以感染进入LX-2细胞。 HBV对Ⅰ型胶原蛋白表达的影响。肝纤维化的主要特征是以Ⅰ型胶原蛋白为主的ECM成分过度沉积，本部分以Ⅰ型胶原α1链为靶标研究HBV致肝纤维化作用。将HBV真核表达载体和空载体分别转染LX-2细胞，48h后收集细胞，Western Blot检测Ⅰ型胶原蛋白表达。结果：HBV表达组Ⅰ型胶原蛋白表达明显高于对照组。这说明在LX-2细胞中，HBV可以促进Ⅰ型胶原蛋白表达。 2. HBV编码蛋白对LX-2细胞Ⅰ型胶原蛋白表达的影响 HBV编码蛋白对COL1A1表达的影响。上述研究中HBV可以促进Ⅰ型胶原蛋白α1链表达，为了探索其分子机制，首先要确定HBV促进Ⅰ型胶原α1链表达的病毒成分。将HBV各编码蛋白真核表达载体和空载体分别转染LX-2细胞，48h后收集细胞。real-time PCR定量检测COL1A1mRNA相对表达，Western Blot检测Ⅰ型胶原蛋白表达。结果表明HBV各编码蛋白不同程度地促进COL1A1mRNA和蛋白表达，其中HBx的作用最强。 HBV编码蛋白对COL1A1启动子活性的影响。前述HBV各编码蛋白均可从转录水平促进COL1A1表达，推测其可能通过上调COL1A1启动子活性促进COL1A1转录表达。将HBV各编码蛋白真核表达载体和空载体分别与COL1A1启动子报告基因载体及内参报告基因载体共转染LX-2细胞，24h后收集细胞，双报告基因检测COL1A1启动子活性。结果证实HBV各编码蛋白不同程度地上调COL1A1启动子活性，其中HBx的作用最强。 3. HBx对LX-2细胞Ⅰ型胶原蛋白表达的调控机制研究 HBV感染LX-2细胞后HBx在细胞中的表达。上述结果发现HBx蛋白可以上调LX-2细胞COL1A1启动子活性进而促进COL1A1表达，且作用强于其它的HBV编码蛋白，以往研究尚无相关结果的报道，因此选择HBx进行深入研究。在研究HBx调控Ⅰ型胶原蛋白表达的机制之前，需要明确HBV感染LX-2细胞后HBx能否在细胞中表达。将LX-2细胞与不同浓度的HBV病毒共培养，48h后去除培养上清,收集并洗涤细胞后裂解，RT-PCR半定量检测HBx mRNA表达，Western Blot检测HBx蛋白表达。结果：随着共培养的HBV浓度升高，HBx mRNA及蛋白表达增加。这说明HBV感染LX-2细胞后HBx蛋白可以在细胞中表达，且其表达量与HBV浓度正相关。 COL1A1启动子上受HBx调控的活性区域。将COL1A1启动子全长分成两段：F1段（-2483～-269bp）和F2段（-268～+42bp），分别构建报告基因载体。将构建的含COL1A1启动子各段及全长报告基因载体分别与内参报告基因载体组合共转染LX-2细胞，双报告基因检测COL1A1启动子各段及全长活性。结果：F2段活性大于F1段活性。将HBx真核表达载体和空载体分别与COL1A1启动子F2段及全长报告基因载体组合转染LX-2细胞，双报告基因检测COL1A1启动子F2段及全长活性。结果：HBx除了促进全长启动子活性，也可以促进COL1A1启动子F2段活性。以上结果说明： COL1A1启动子F2段（-268～+42bp）是受HBx调控的活性区域，这为进一步研究其调控通路提供了依据。 HBx调控COL1A1表达过程中TGFβ1的作用。TGFβ1在HSC的活化及COL1A1表达过程中发挥重要作用。为了研究TGFβ1是否在HBx调控COL1A1表达过程中起作用，使用定量PCR检测HBx是否促进TGFβ1的表达，双报告基因法检测抑制TGFβ1的表达是否影响HBx对COL1A1启动子活性的上调作用。结果：HBx不促进TGFβ1的表达；抑制TGFβ1的表达不影响HBx对COL1A1启动子活性的上调作用。这说明TGFβ1不参与HBx对Ⅰ型胶原蛋白表达的调控。贾继东等研究结果：用TGFβ1中和抗体阻断TGFβ1与受体的结合不影响HBV对LX-2细胞Ⅰ型胶原蛋白表达的促进作用。这与本研究的结果一致。 HBx调控COL1A1表达过程中P38MAPK通路的作用。P38MAPK通路在HBV诱导的COL1A1表达过程中发挥作用。为了研究HBx对COL1A1启动子的上调作用是否通过P38MAPK通路实现，将HBx真核表达载体和空载体分别与COL1A1启动子报告基因载体及内参报告基因载体共转染LX-2细胞，6h后，加P38MAPK通路阻断剂SB203580，24h后收集细胞，双报告基因检测COL1A1启动子活性。结果：阻断P38MAPK通路后，HBx对COL1A1启动子活性的上调作用被抑制。这说明P38MAPK通路参与HBx对Ⅰ型胶原蛋白表达的调控。二、新型抗纤维化药物筛选模型研究肝纤维化的主要特征是以Ⅰ型胶原蛋白为主的ECM成分过度沉积，故抑制Ⅰ型胶原表达是目前抗肝纤维化治疗的重要策略之一。转录水平基因启动子活性的调节是基因表达调控的关键环节之一，本课题第一部分的研究结果也能很好的说明这一点，即COL1A1mRNA及蛋白表达与COL1A1启动子活性具有很好的对应关系。本研究第二部分拟以COL1A1启动子报告基因系统为基础建立抑制Ⅰ型胶原蛋白表达的抗纤维化药物筛选和评价模型。 1.抗纤维化药物筛选模型的建立 COL1A1启动子报告基因载体的构建。根据COL1A1基因上游的序列设计合成扩增引物，以人基因组DNA为模板，PCR扩增COL1A1启动子序列，将其插入报告基因载体pGL4中，构建COL1A1启动子报告基因载体pGL4-COL1A1promoter，限制性酶切和DNA测序鉴定其正确性。抗纤维化药物筛选模型的建立及检测。以LX-2细胞为宿主细胞，将COL1A1启动子报告基因载体与内参报告基因载体共转染LX-2细胞，24h后收集细胞，双报告基因检测COL1A1启动子活性。抗纤维化药物筛选模型建立之后，使用对Ⅰ型胶原蛋白表达有抑制作用的TGFβ1siRNA验证其有效性。结果：TGFβ1siRNA不影响COL1A1启动子活性。分析其原因：一方面可能与细胞内源性TGFβ1表达低下有关；另一方面与正常LX-2细胞COL1A1启动子处于低活化状态有关。拟外源表达活化剂TGFβ1活化LX-2细胞，模拟肝纤维化肝星状细胞的高活化状态，再进行药物评价实验。 2.抗纤维化药物筛选模型的优化抗纤维化药物筛选模型的首次优化。以HepG2细胞提取的总RNA逆转录产物为模板，PCR扩增TGFβ1全长编码基因，插入到表达载体pcDNA3.1中，构建TGFβ1的重组载体pcDNA3.1-TGFβ1，限制性酶切和DNA测序鉴定为正确连接的克隆。将pcDNA3.1、pcDNA3.1-TGFβ1分别与COL1A1启动子报告基因载体及内参报告基因载体共转染LX-2细胞，24h后收集细胞，双报告基因检测COL1A1启动子活性。结果：过表达TGFβ1仅使COL1A1启动子活性提高了不到2倍（t=3.396，P=0.0274）。推测其原因：可能是TGFβ1在细胞内表达但没有分泌到细胞外，无法与细胞膜受体结合进而发挥作用。拟构建含分泌信号肽的TGFβ1重组表达载体，验证上述推测是否正确。抗纤维化药物筛选模型的再次优化。同上方法构建含分泌信号肽的TGFβ1重组表达载体pJW4303-TGFβ1。将pJW4303、pJW4303-TGFβ1分别与COL1A1启动子报告基因载体及内参报告基因载体共转染LX-2细胞，24h后收集细胞，双报告基因检测COL1A1启动子活性。结果：分泌表达TGFβ1使COL1A1启动子活性提高了200倍以上（t=21.78，P=0.0001）。该结果验证了上述推测：TGFβ1只有被分泌到细胞外并与自身细胞膜受体结合后才能发挥活化作用。故选择pJW4303-TGFβ1用于抗纤维化药物筛选模型的优化。 3.抗纤维化药物筛选模型的有效性鉴定文献报道糖皮质激素类药物地塞米松可以下调COL1A1启动子活性进而抑制Ⅰ型胶原蛋白表达，故选其作为阳性模式药物来鉴定该筛选模型的有效性。在高活化状态的筛选模型中，加入不同浓度地塞米松，观察其抑制效果。结果：地塞米松对COL1A1启动子活性具有明显的抑制作用且具有剂量依赖关系。这说明该模型可以用于筛选和评价抑制I型胶原蛋白表达的抗纤维化药物。本研究证实了HBV能感染LX-2细胞并促进Ⅰ型胶原蛋白表达；HBV编码蛋白尤其是HBx能上调COL1A1启动子活性进而促进Ⅰ型胶原蛋白表达；TGFβ1不参与而P38MAPK通路参与HBx对Ⅰ型胶原蛋白的调控。另外，本研究以COL1A1启动子活性调控机制为基础，建立了一种新型抗纤维化药物筛选模型，为抑制COL1A1启动子活性进而抑制Ⅰ型胶原蛋白表达的抗纤维化药物研究奠定了基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62;R575.2

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