1. 重要細胞因子對肝前體細胞誘導分化實驗 2. 丙型肝炎病毒F蛋白的原核表達與腺病毒構(gòu)建
發(fā)布時間：2018-09-09 15:46
【摘要】： 目的:目前臨床治療肝癌,肝硬化的主要手段是肝移植,但由于肝供體缺乏,手術(shù)風險大,費用高,易發(fā)生免疫排斥反應等原因制約了這一技術(shù)的發(fā)展,因此以肝細胞移植和生物人工肝為代表的肝細胞替代治療,逐漸成為終末期肝病治療研究的熱點。而尋找合適的肝細胞來源則是肝細胞移植在臨床上廣泛開展的緊迫問題。 肝前體細胞(Hepatic Progenitor cells, HP)直接參與肝臟的生長和發(fā)育,同時也是肝細胞移植和細胞再生的重要來源。其表型特征更接近肝細胞,在研究肝細胞的發(fā)育和分化、信號調(diào)控機理、肝細胞生物學以及病毒性肝炎發(fā)病機理和抗病毒藥物篩選研究上是比胚胎干細胞更理想的細胞來源。 本項目擬建立永生化的肝前體細胞克隆,為深入研究肝干細胞定向分化機制提供理想的實驗材料,進一步在該細胞模型上篩選并優(yōu)化高效的肝細胞定向分化誘導方法,探討肝干細胞向肝細胞特異分化過程及分子機制,并著重進行HGF、LIF和BMP信號誘導肝前體細胞分化的實驗研究。本項目不僅對解析肝臟發(fā)育機理、探索肝細胞定向分化具有重要的理論價值,并且可以通過人為調(diào)節(jié)肝細胞分化來獲取充足和具備生物學功能的成熟肝細胞,為肝細胞移植、肝組織學工程、生物人工肝等治療方式提供有效的細胞來源,具有潛在的臨床應用前景。 方法:運用LoxP位點特異修飾的SV40T逆轉(zhuǎn)錄病毒SSR#69導入胚胎肝細胞,建立永生化肝前體細胞,篩選和鑒定單克隆肝前體細胞株。體外誘導分化實驗分別采用含人LIF、BMP2、BMP9基因的重組腺病毒感染肝前體細胞,在病毒感染后第4天、7天和10天用糖原染色和ICG攝取實驗觀察肝前體細胞的分化成熟度,并在第5、7、10天通過檢測白蛋白啟動子調(diào)控的熒光素酶報告基因活性,觀察細胞誘導合成白蛋白情況。 結(jié)果:成功建立了永生化肝前體細胞株,為體外觀察肝前體細胞的誘導分化建立了良好的細胞模型。篩選并獲得了4株單克隆肝前體細胞株,分別為HP13-6,13-19,14-2和14-19。其中HP13-6和14-19細胞形態(tài)均一,肝特異性基因的表達最強。體外誘導實驗結(jié)果表明,BMP2和BMP9對HP14-19的誘導作用最強, PAS染色和ICG攝取細胞陽性率隨誘導時間的延長明顯上升,誘導后第7天效果最明顯。PAS染色陽性細胞率BMP2和BMP9分別為30%和45%;ICG細胞陽性率BMP2和BMP9分別為40%和30%,熒光素酶活性普遍在誘導后第10天達高峰,HP13-6細胞對BMP9的誘導應答最強,酶活性增加了近10倍,其次為LIF(8倍),BMP2(7倍)和HGF(6倍);HP14-19則對BMP2的應答效果最佳,酶活性增加了7倍,其次為LIF(5倍),HGF(2.5倍)。LIF誘導HP14-19后,PAS染色和ICG攝取細胞陽性率略為增加,熒光素酶活性有一定變化,提示LIF對HP14-19誘導作用不明顯。 結(jié)論:建立永生化的肝前體細胞模型,篩選并獲得了4株具有代表性的單克隆肝前體細胞株,BMP2和BMP9能夠誘導HP14-19向發(fā)育晚期肝細胞分化,并初步具備成熟肝細胞的一些功能。 目的:丙型肝炎病毒(Hepatitis C Virus,HCV)呈世界范圍內(nèi)流行,全球大約有1.8億人被感染,感染丙型肝炎病毒是發(fā)生肝癌的一個重要因素,大約2-5%的HCV感染者最終發(fā)展成肝癌。迄今為止,HCV的疫苗尚未解決,干擾素加利巴韋林是治療丙型肝炎的主要方法,但其最終療效還不能明確。丙型肝炎病毒是單鏈正股RNA病毒,長約9.6Kb,編碼由3000個氨基酸殘基組成的多氨酸。蛋白前體最后加工成10個成熟的蛋白: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譯過程中核糖體發(fā)生移位產(chǎn)生的,但目前對它的生物學性質(zhì)還不了解,F蛋白在肝癌的發(fā)生中起了什么作用?F蛋白對細胞的影響是什么?這些都是值得深入研究的問題。本課題構(gòu)建F蛋白的原核表達載體并純化F蛋白,有利于下一步單克隆抗體的制備,構(gòu)建F蛋白的腺病毒載體,為深入研究F蛋白的生物學功能奠定了基礎(chǔ)。 方法:本研究內(nèi)容分為兩部分,第一部分以pET32a(+)為載體,構(gòu)建F蛋白的原核表達質(zhì)粒,并在大腸桿菌BL21中表達F蛋白。主要技術(shù)路線為:根據(jù)丙型肝炎病毒F基因的全序列設(shè)計引物,以HCV1a型cDNA質(zhì)粒H/FL為模板,通過PCR方法擴增得到F基因的編碼區(qū)序列,將其定向克隆于含有6×His標簽的原核表達載體pET32a(+),轉(zhuǎn)化大腸桿菌克隆菌株JM109,經(jīng)菌落PCR篩選,雙酶切和測序鑒定陽性克隆后,再次轉(zhuǎn)化大腸桿菌表達菌BL21后經(jīng)IPTG誘導,出現(xiàn)了與預期分子量相符的蛋白條帶。表達產(chǎn)物經(jīng)SDS-PAGE電泳分析和Western-blot檢測鑒定,并用Ni-NTA親和層析柱純化。第二部分,構(gòu)建含有HCV F蛋白重組腺病毒載體,經(jīng)與腺病毒骨架質(zhì)粒pAdEasy-1在BJ5183同源重組后轉(zhuǎn)染HEK293細胞,包裝成腺病毒并經(jīng)多輪擴增,得到高滴度的感染病毒。 結(jié)果:F基因以正確的方式插入到pET32a(+)載體中,重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21后經(jīng)IPTG誘導表達,出現(xiàn)了與預期分子量相符的蛋白條帶,進一步通過Western blot證實成功表達了含有6×His tag蛋白的融合蛋白。測序及酶切鑒定結(jié)果表明:目的基因正確插入腺病毒穿梭質(zhì)粒,轉(zhuǎn)染HEK293細胞后在熒光顯微鏡下觀察,細胞中含有綠色熒光。 結(jié)論:成功構(gòu)建了丙型肝炎病毒F蛋白的原核表達載體pET32a(+)-HCVF并表達和純化重組融合蛋白,構(gòu)建了含有F基因的腺病毒載體,得到高滴度的腺病毒,為進一步研究F蛋白的生物學功能奠定了基礎(chǔ)。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R575;R373.21
本文編號：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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R575;R373.21
【參考文獻】
相關(guān)期刊論文 前5條
1 呂國悅;張平;李紅;李爽;王廣義;;人成體肝臟干細胞體外分離、培養(yǎng)及鑒定[J];吉林大學學報(醫(yī)學版);2007年02期
2 劉善榮;Oct4調(diào)節(jié)胚胎干細胞全能性擴增的研究進展[J];第二軍醫(yī)大學學報;2003年09期
3 黃小兵,梁平;細胞因子對肝干細胞分化調(diào)節(jié)的研究進展[J];國外醫(yī)學(消化系疾病分冊);2005年01期
4 武文斌;邵圣文;于建國;戚中田;;丙型肝炎病毒F蛋白對p21基因的調(diào)節(jié)作用[J];熱帶醫(yī)學雜志;2007年09期
5 呂曉輝;曹人月;佟靜;宋敏;;STAT3在肝細胞生長因子促進大鼠WB細胞增生過程中的作用[J];山西醫(yī)藥雜志;2008年07期
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