1. 重要细胞因子对肝前体细胞诱导分化实验 2. 丙型肝炎病毒F蛋白的原核表达与腺病毒构建
发布时间：2018-09-09 15:46
【摘要】： 目的:目前临床治疗肝癌,肝硬化的主要手段是肝移植,但由于肝供体缺乏,手术风险大,费用高,易发生免疫排斥反应等原因制约了这一技术的发展,因此以肝细胞移植和生物人工肝为代表的肝细胞替代治疗,逐渐成为终末期肝病治疗研究的热点。而寻找合适的肝细胞来源则是肝细胞移植在临床上广泛开展的紧迫问题。 肝前体细胞(Hepatic Progenitor cells, HP)直接参与肝脏的生长和发育,同时也是肝细胞移植和细胞再生的重要来源。其表型特征更接近肝细胞,在研究肝细胞的发育和分化、信号调控机理、肝细胞生物学以及病毒性肝炎发病机理和抗病毒药物筛选研究上是比胚胎干细胞更理想的细胞来源。 本项目拟建立永生化的肝前体细胞克隆,为深入研究肝干细胞定向分化机制提供理想的实验材料,进一步在该细胞模型上筛选并优化高效的肝细胞定向分化诱导方法,探讨肝干细胞向肝细胞特异分化过程及分子机制,并着重进行HGF、LIF和BMP信号诱导肝前体细胞分化的实验研究。本项目不仅对解析肝脏发育机理、探索肝细胞定向分化具有重要的理论价值,并且可以通过人为调节肝细胞分化来获取充足和具备生物学功能的成熟肝细胞,为肝细胞移植、肝组织学工程、生物人工肝等治疗方式提供有效的细胞来源,具有潜在的临床应用前景。 方法:运用LoxP位点特异修饰的SV40T逆转录病毒SSR#69导入胚胎肝细胞,建立永生化肝前体细胞,筛选和鉴定单克隆肝前体细胞株。体外诱导分化实验分别采用含人LIF、BMP2、BMP9基因的重组腺病毒感染肝前体细胞,在病毒感染后第4天、7天和10天用糖原染色和ICG摄取实验观察肝前体细胞的分化成熟度,并在第5、7、10天通过检测白蛋白启动子调控的荧光素酶报告基因活性,观察细胞诱导合成白蛋白情况。 结果:成功建立了永生化肝前体细胞株,为体外观察肝前体细胞的诱导分化建立了良好的细胞模型。筛选并获得了4株单克隆肝前体细胞株,分别为HP13-6,13-19,14-2和14-19。其中HP13-6和14-19细胞形态均一,肝特异性基因的表达最强。体外诱导实验结果表明,BMP2和BMP9对HP14-19的诱导作用最强, PAS染色和ICG摄取细胞阳性率随诱导时间的延长明显上升,诱导后第7天效果最明显。PAS染色阳性细胞率BMP2和BMP9分别为30%和45%;ICG细胞阳性率BMP2和BMP9分别为40%和30%,荧光素酶活性普遍在诱导后第10天达高峰,HP13-6细胞对BMP9的诱导应答最强,酶活性增加了近10倍,其次为LIF(8倍),BMP2(7倍)和HGF(6倍);HP14-19则对BMP2的应答效果最佳,酶活性增加了7倍,其次为LIF(5倍),HGF(2.5倍)。LIF诱导HP14-19后,PAS染色和ICG摄取细胞阳性率略为增加,荧光素酶活性有一定变化,提示LIF对HP14-19诱导作用不明显。 结论:建立永生化的肝前体细胞模型,筛选并获得了4株具有代表性的单克隆肝前体细胞株,BMP2和BMP9能够诱导HP14-19向发育晚期肝细胞分化,并初步具备成熟肝细胞的一些功能。 目的:丙型肝炎病毒(Hepatitis C Virus,HCV)呈世界范围内流行,全球大约有1.8亿人被感染,感染丙型肝炎病毒是发生肝癌的一个重要因素,大约2-5%的HCV感染者最终发展成肝癌。迄今为止,HCV的疫苗尚未解决,干扰素加利巴韦林是治疗丙型肝炎的主要方法,但其最终疗效还不能明确。丙型肝炎病毒是单链正股RNA病毒,长约9.6Kb,编码由3000个氨基酸残基组成的多氨酸。蛋白前体最后加工成10个成熟的蛋白:核心蛋白(core)、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B。 F蛋白是核心蛋白在翻译过程中核糖体发生移位产生的,但目前对它的生物学性质还不了解,F蛋白在肝癌的发生中起了什么作用?F蛋白对细胞的影响是什么?这些都是值得深入研究的问题。本课题构建F蛋白的原核表达载体并纯化F蛋白,有利于下一步单克隆抗体的制备,构建F蛋白的腺病毒载体,为深入研究F蛋白的生物学功能奠定了基础。 方法:本研究内容分为两部分,第一部分以pET32a(+)为载体,构建F蛋白的原核表达质粒,并在大肠杆菌BL21中表达F蛋白。主要技术路线为:根据丙型肝炎病毒F基因的全序列设计引物,以HCV1a型cDNA质粒H/FL为模板,通过PCR方法扩增得到F基因的编码区序列,将其定向克隆于含有6×His标签的原核表达载体pET32a(+),转化大肠杆菌克隆菌株JM109,经菌落PCR筛选,双酶切和测序鉴定阳性克隆后,再次转化大肠杆菌表达菌BL21后经IPTG诱导,出现了与预期分子量相符的蛋白条带。表达产物经SDS-PAGE电泳分析和Western-blot检测鉴定,并用Ni-NTA亲和层析柱纯化。第二部分,构建含有HCV F蛋白重组腺病毒载体,经与腺病毒骨架质粒pAdEasy-1在BJ5183同源重组后转染HEK293细胞,包装成腺病毒并经多轮扩增,得到高滴度的感染病毒。 结果:F基因以正确的方式插入到pET32a(+)载体中,重组质粒转化大肠杆菌BL21后经IPTG诱导表达,出现了与预期分子量相符的蛋白条带,进一步通过Western blot证实成功表达了含有6×His tag蛋白的融合蛋白。测序及酶切鉴定结果表明:目的基因正确插入腺病毒穿梭质粒,转染HEK293细胞后在荧光显微镜下观察,细胞中含有绿色荧光。 结论:成功构建了丙型肝炎病毒F蛋白的原核表达载体pET32a(+)-HCVF并表达和纯化重组融合蛋白,构建了含有F基因的腺病毒载体,得到高滴度的腺病毒,为进一步研究F蛋白的生物学功能奠定了基础。
[Abstract]:OBJECTIVE: At present, liver transplantation is the main method for the treatment of liver cancer and liver cirrhosis. However, due to the lack of liver donors, high risk of operation, high cost and easy occurrence of immune rejection, the development of this technology is restricted. Therefore, hepatocyte replacement therapy, represented by hepatocyte transplantation and bioartificial liver, has gradually become the research and treatment of end-stage liver disease. It is an urgent problem to find a suitable source of hepatocytes for hepatocyte transplantation.
Hepatic Progenitor Cells (HP) are directly involved in the growth and development of the liver, and are also an important source of liver cell transplantation and cell regeneration. Screening studies are more ideal cell sources than embryonic stem cells.
The aim of this project is to establish an immortalized hepatic precursor cell clone to provide an ideal experimental material for further study on the mechanism of directional differentiation of hepatic stem cells, to screen and optimize the efficient induction method of directional differentiation of hepatic cells on this cell model, to explore the process and molecular mechanism of hepatic stem cells differentiating into hepatocytes, and to focus on HGF, LIF. This project not only has important theoretical value to analyze the mechanism of liver development and explore the directional differentiation of hepatocytes, but also can obtain sufficient and biologically functional mature hepatocytes by regulating the differentiation of hepatocytes artificially. Artificial liver and other treatment methods provide effective cell sources and have potential clinical application prospects.
METHODS: Immortalized hepatic precursor cells were established by introducing LoxP site-specific retrovirus SSR # 69 into embryonic hepatocytes. Monoclonal hepatic precursor cell lines were screened and identified. The differentiation and maturation of hepatic precursor cells were observed by glycogen staining and ICG uptake assay. The activity of luciferase reporter gene regulated by albumin promoter was detected on the 5th, 7th and 10th day.
RESULTS: Immortalized hepatic precursor cell lines were successfully established, and a good cell model was established to observe the differentiation of hepatic precursor cells in vitro. Four monoclonal hepatic precursor cell lines, HP13-6, 13-19, 14-2 and 14-19, were screened and obtained, respectively. The results showed that BMP2 and BMP9 had the strongest induction effect on HP14-19, and the positive rates of PAS staining and ICG uptake cells increased significantly with the prolongation of induction time. The positive rates of BMP2 and BMP9 were 30% and 45% respectively. The positive rates of BMP2 and BMP9 in ICG cells were 40% and 30%, respectively. Luciferase activity was generally induced. On the 10th day after induction, the response of HP13-6 cells to BMP9 was the strongest, and the enzyme activity increased nearly 10 times, followed by LIF (8 times), BMP2 (7 times) and HGF (6 times); HP14-19 cells showed the best response to BMP2, with the enzyme activity increased 7 times, followed by LIF (5 times), HGF (2.5 times). After induction of HP14-19 by LIF, the positive rate of PAS staining and ICG uptake cells slightly increased, fluorescence rate increased. There was a certain change in the activity of photo enzyme, suggesting that LIF had no obvious effect on HP14-19 induction.
CONCLUSION: Four representative monoclonal hepatic precursor cell lines were screened and obtained by establishing immortalized hepatic precursor cell model. BMP2 and BMP9 could induce HP14-19 to differentiate into advanced hepatocytes and possess some functions of mature hepatocytes.
Objective: Hepatitis C Virus (HCV) is a worldwide epidemic. About 180 million people are infected worldwide. Hepatitis C virus infection is an important factor in the development of hepatocellular carcinoma. About 2-5% of people infected with HCV eventually develop into hepatocellular carcinoma. Hepatitis C virus is a single-stranded positive-stranded RNA virus, about 9.6Kb long, encoding a polypeptide of 3000 amino acid residues. Protein precursors are finally processed into 10 mature proteins: core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.
F protein is produced by ribosome translocation in the process of translation, but its biological properties are still unknown. What role does F protein play in the development of hepatocellular carcinoma? What is the effect of F protein on cells? These are all questions worthy of further study. It is helpful for the preparation of monoclonal antibody and the construction of adenovirus vector of F protein, which lays a foundation for the further study of biological function of F protein.
Methods: In the first part, the prokaryotic expression plasmid of F protein was constructed with pET32a (+) as the vector, and F protein was expressed in E. coli BL21. The recombinant plasmid was cloned into prokaryotic expression vector pET32a (+) containing 6 *His tag and transformed into E. coli strain JM109. The positive clones were screened by colony PCR, identified by double enzyme digestion and sequencing, and transformed into E. coli expression strain BL21 again. After induction by IPTG, a protein band with expected molecular weight was found. The recombinant adenovirus vector containing HCV F protein was constructed. The recombinant adenovirus vector was transfected into HEK293 cells after homologous recombination with adenovirus cytoskeleton plasmid pAdEasy-1 in BJ5183. The recombinant adenovirus was packaged into adenovirus and amplified in multiple rounds.
Results: The F gene was inserted into the pET32a (+) vector in the correct way. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. A protein band with expected molecular weight was found. The fusion protein containing 6 *His tag protein was confirmed by Western blot. The adenovirus shuttle plasmid was inserted correctly and transfected into HEK293 cells. The cells showed green fluorescence under fluorescence microscope.
CONCLUSION: The prokaryotic expression vector pET32a (+) - HCVF of hepatitis C virus F protein was successfully constructed, and the recombinant fusion protein was expressed and purified. The adenovirus vector containing F gene was constructed, and the high titer adenovirus was obtained, which laid a foundation for further study of the biological function of F protein.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R575;R373.21
本文编号：2232816
[Abstract]:OBJECTIVE: At present, liver transplantation is the main method for the treatment of liver cancer and liver cirrhosis. However, due to the lack of liver donors, high risk of operation, high cost and easy occurrence of immune rejection, the development of this technology is restricted. Therefore, hepatocyte replacement therapy, represented by hepatocyte transplantation and bioartificial liver, has gradually become the research and treatment of end-stage liver disease. It is an urgent problem to find a suitable source of hepatocytes for hepatocyte transplantation.
Hepatic Progenitor Cells (HP) are directly involved in the growth and development of the liver, and are also an important source of liver cell transplantation and cell regeneration. Screening studies are more ideal cell sources than embryonic stem cells.
The aim of this project is to establish an immortalized hepatic precursor cell clone to provide an ideal experimental material for further study on the mechanism of directional differentiation of hepatic stem cells, to screen and optimize the efficient induction method of directional differentiation of hepatic cells on this cell model, to explore the process and molecular mechanism of hepatic stem cells differentiating into hepatocytes, and to focus on HGF, LIF. This project not only has important theoretical value to analyze the mechanism of liver development and explore the directional differentiation of hepatocytes, but also can obtain sufficient and biologically functional mature hepatocytes by regulating the differentiation of hepatocytes artificially. Artificial liver and other treatment methods provide effective cell sources and have potential clinical application prospects.
METHODS: Immortalized hepatic precursor cells were established by introducing LoxP site-specific retrovirus SSR # 69 into embryonic hepatocytes. Monoclonal hepatic precursor cell lines were screened and identified. The differentiation and maturation of hepatic precursor cells were observed by glycogen staining and ICG uptake assay. The activity of luciferase reporter gene regulated by albumin promoter was detected on the 5th, 7th and 10th day.
RESULTS: Immortalized hepatic precursor cell lines were successfully established, and a good cell model was established to observe the differentiation of hepatic precursor cells in vitro. Four monoclonal hepatic precursor cell lines, HP13-6, 13-19, 14-2 and 14-19, were screened and obtained, respectively. The results showed that BMP2 and BMP9 had the strongest induction effect on HP14-19, and the positive rates of PAS staining and ICG uptake cells increased significantly with the prolongation of induction time. The positive rates of BMP2 and BMP9 were 30% and 45% respectively. The positive rates of BMP2 and BMP9 in ICG cells were 40% and 30%, respectively. Luciferase activity was generally induced. On the 10th day after induction, the response of HP13-6 cells to BMP9 was the strongest, and the enzyme activity increased nearly 10 times, followed by LIF (8 times), BMP2 (7 times) and HGF (6 times); HP14-19 cells showed the best response to BMP2, with the enzyme activity increased 7 times, followed by LIF (5 times), HGF (2.5 times). After induction of HP14-19 by LIF, the positive rate of PAS staining and ICG uptake cells slightly increased, fluorescence rate increased. There was a certain change in the activity of photo enzyme, suggesting that LIF had no obvious effect on HP14-19 induction.
CONCLUSION: Four representative monoclonal hepatic precursor cell lines were screened and obtained by establishing immortalized hepatic precursor cell model. BMP2 and BMP9 could induce HP14-19 to differentiate into advanced hepatocytes and possess some functions of mature hepatocytes.
Objective: Hepatitis C Virus (HCV) is a worldwide epidemic. About 180 million people are infected worldwide. Hepatitis C virus infection is an important factor in the development of hepatocellular carcinoma. About 2-5% of people infected with HCV eventually develop into hepatocellular carcinoma. Hepatitis C virus is a single-stranded positive-stranded RNA virus, about 9.6Kb long, encoding a polypeptide of 3000 amino acid residues. Protein precursors are finally processed into 10 mature proteins: core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.
F protein is produced by ribosome translocation in the process of translation, but its biological properties are still unknown. What role does F protein play in the development of hepatocellular carcinoma? What is the effect of F protein on cells? These are all questions worthy of further study. It is helpful for the preparation of monoclonal antibody and the construction of adenovirus vector of F protein, which lays a foundation for the further study of biological function of F protein.
Methods: In the first part, the prokaryotic expression plasmid of F protein was constructed with pET32a (+) as the vector, and F protein was expressed in E. coli BL21. The recombinant plasmid was cloned into prokaryotic expression vector pET32a (+) containing 6 *His tag and transformed into E. coli strain JM109. The positive clones were screened by colony PCR, identified by double enzyme digestion and sequencing, and transformed into E. coli expression strain BL21 again. After induction by IPTG, a protein band with expected molecular weight was found. The recombinant adenovirus vector containing HCV F protein was constructed. The recombinant adenovirus vector was transfected into HEK293 cells after homologous recombination with adenovirus cytoskeleton plasmid pAdEasy-1 in BJ5183. The recombinant adenovirus was packaged into adenovirus and amplified in multiple rounds.
Results: The F gene was inserted into the pET32a (+) vector in the correct way. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. A protein band with expected molecular weight was found. The fusion protein containing 6 *His tag protein was confirmed by Western blot. The adenovirus shuttle plasmid was inserted correctly and transfected into HEK293 cells. The cells showed green fluorescence under fluorescence microscope.
CONCLUSION: The prokaryotic expression vector pET32a (+) - HCVF of hepatitis C virus F protein was successfully constructed, and the recombinant fusion protein was expressed and purified. The adenovirus vector containing F gene was constructed, and the high titer adenovirus was obtained, which laid a foundation for further study of the biological function of F protein.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R575;R373.21
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