胎儿肝干细胞诱导分化的实验研究
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摘要
背景 目前,临床上各种原因引起的急、慢性肝功能衰竭的发病率和死亡率仍然很高,原位肝移植(orthotopiC liver transplantation,OLT)是治疗肝功能衰竭的有效方法,但供体短缺、费用昂贵及免疫排斥等因素限制了其广泛应用。生物型人工肝(Bioartificial liver,BAL)和肝细胞移植(hepatocyte transplantation,HCT)可以暂时替代衰竭肝脏的功能,其疗效在各种动物实验及少量临床试验中得到证实,这两种支持治疗可使患者肝脏得到再生或者桥接肝移植,它们在临床应用中具有很好的前景。但是,限制生物型人工肝和肝细胞移植广泛应用的关键问题是没有合适的细胞来源。
近年来,随着干细胞研究的理论和技术不断深入,研究者试图由干细胞诱导分化出成熟肝细胞,进而为生物型人工肝和肝细胞移植的临床应用提供细胞来源。其中,肝干细胞是一种成体多能干细胞,它具有自我更新、高度增殖及向肝细胞等多种组织分化的能力,若能将其大量扩增再诱导分化为肝细胞,就可能从量和质上解决上述细胞来源短缺的问题。所以,肝干细胞在生物医学工程和组织工程领域具有潜在应用价值,为人工培养大量有用的肝细胞并用于临床带来了希望。
肝干细胞的研究尚处于基础研究阶段,研究对象多选用大鼠、小鼠等动物模型作为实验材料。可以肯定的结论是肝脏中存在肝干细胞,它在体内可以分化为肝细胞和胆管细胞,参与肝脏的发生发育及损伤后再生等生理病理过程,在体外培养时仍具有很强的增殖和分化能力。这些动物实验有助于阐明肝干细胞的发生及体内外增殖分化等生物学特性,但将来用于临床治疗的肝干细胞以人源性的细胞最为合适,这样可以避免动物源性细胞所带来的未知病原体感染和异种基因导入人体的问题,细胞的安全性更高,并且一些实验证实正常成体肝脏中细胞组成相对稳定,肝干细胞含量很少且多处于休眠
Background At present the mortality of acute or chronic hepatic failure is still very high and the effective treatment is orthotopic liver transplantation(OLT).But the extensive application of OLT is restricted due to clinical shortage of donor organs. BAL(Bioartif icial liver)and HCT(Hepatocyte transplantation) can replace the failed liver temporarily. Researchers have acquired some successful experience from various animal model experiments, however, most of researchers pay more attention to heterogenic animal hepatocytes. But these heterogenic hepatocytes maybe bring the infection of unknown pathogens and spread of heterogen in the crowd. If cell resource is adequate, BAL and HCT will have a good prospect in clinical application. Recently hepatic stem cells has become a hot topic in the world. The hepatic stem cells are considered to be a kind of bipotential cells which can differentiate into hepatocytes and biliary epithelial cells. If we can proliferate hepatic stem cells and differentiate them into mature hepatocytes, we will solve above-mentioned problems about cell source. In this experiment we want to prove whether the human fetal HSCs can be induced and differentiated into hepatocytes and be used in the future. We will make some preparations for the clinical application of hepatic stem cells.Part I Isolation and culture of human fetal hepatic stem cells
Objective To establish a way of isolation, purification, identification and culture of human fetal hepatic stem cells. Methods Liver cells were isolated by collagenase (type IV) digestion method, and hepatic stem cells were purified from liver cells by Percoll discontinuous gradient centrifugat ion. Human fetal HSCs were obtained from the liver of aborted fetus in the second trimester of pregnancy. Under the asepsis conditions, establish umbilical-inferior vena extrinsic circulation. Perfuse EGTA balance liquid and collegen solution one by one. Then filtrate liver tissue by filter net and gain liver cells suspension. Centrifugate the liver cells suspension in three kinds of Percoll work solutions with different densities(1. llOg/ml, 1. 070g/ml, 1. 035g/ml). Extract the cells (HSCs) between the middle layer of Percoll and the bottom one. Then adjust the density of cells and culture them in DMEM/F12 media.The viability of hepatic stem cells was checked by trypan blue excluded test. The morphologic characters of human HSCs were observed by optic and electronic microscopy. The surface markers of HSCs including albumin, CK19, AFP and CD34 were identified by immuno- cytochemical staining. Count the amount of the cells during the whole process. Compute the yield of liver cells and human HSCs respectively. Results After two-step collagenase purfusion and centrifugation in discontinuous dentity Percoll solution, the HSCs could be purified from liver cells. The mean viability of HSCs is (88.35 + 2.45)% by trypan blue excluded test. By optic microscopy we can observe that HSCs have oval appearance and are smaller than mature hepatocytes. By electronic microscopy we can find that the diameter of HSCs is about 10 in, there are some microvilli on their surface, the oval nucleus occupy the most
space of the cell and there are few cytoplasm, endoplasmic reticula, mitochondria and other organelle. Immunocytochemical staining shows that the HSCs present the positive signal of CK19, CD34 and AFP and the negative signal of ALB. During short-term culture course the HSCs gradually adhere and begin to grow like epithelia. The mean yield of liver cells is (8. 72 + 1. 02) XlO'/liver (n = 15) , the mean yield of HSCs is (2.34+0.93) XlOVliver (n=15) or (8. 76+0. 46) X105yh/g (n=15) .Conclusions Human HSCs can be obtained by purfusion of collagenase solution and centrifugation of discontinuous dentity Percoll solution satisfactorily. We can acquire enough HSCs with considerable viability and use them for the following experiment.Part II Inducement and differentiation of human fetal hepaticstem cells with HGF and EGF in conditional culture mediaObjective Induce and differentiate human fetal HSCs into hepatocytes with HGF and EGF in conditional culture media. Methods Culture the HSCs in DMEM/F12 media with appropriate cell density as soon as they have been isolated. By MTT colorimetry observe the dose-effect relation of HGF or EGF in five groups which have different concentration of HGF or EGF except for one control group. In inducement experiment we devicle four groups, namely, negative control group, HGF group, EGF group and HGF+EGF group. Every three days replace the media and collect the specimens to be measured. Observe cells by optic microscopy and depict the growth-curves. Measure the content of urea
近年来,随着干细胞研究的理论和技术不断深入,研究者试图由干细胞诱导分化出成熟肝细胞,进而为生物型人工肝和肝细胞移植的临床应用提供细胞来源。其中,肝干细胞是一种成体多能干细胞,它具有自我更新、高度增殖及向肝细胞等多种组织分化的能力,若能将其大量扩增再诱导分化为肝细胞,就可能从量和质上解决上述细胞来源短缺的问题。所以,肝干细胞在生物医学工程和组织工程领域具有潜在应用价值,为人工培养大量有用的肝细胞并用于临床带来了希望。
肝干细胞的研究尚处于基础研究阶段,研究对象多选用大鼠、小鼠等动物模型作为实验材料。可以肯定的结论是肝脏中存在肝干细胞,它在体内可以分化为肝细胞和胆管细胞,参与肝脏的发生发育及损伤后再生等生理病理过程,在体外培养时仍具有很强的增殖和分化能力。这些动物实验有助于阐明肝干细胞的发生及体内外增殖分化等生物学特性,但将来用于临床治疗的肝干细胞以人源性的细胞最为合适,这样可以避免动物源性细胞所带来的未知病原体感染和异种基因导入人体的问题,细胞的安全性更高,并且一些实验证实正常成体肝脏中细胞组成相对稳定,肝干细胞含量很少且多处于休眠
Background At present the mortality of acute or chronic hepatic failure is still very high and the effective treatment is orthotopic liver transplantation(OLT).But the extensive application of OLT is restricted due to clinical shortage of donor organs. BAL(Bioartif icial liver)and HCT(Hepatocyte transplantation) can replace the failed liver temporarily. Researchers have acquired some successful experience from various animal model experiments, however, most of researchers pay more attention to heterogenic animal hepatocytes. But these heterogenic hepatocytes maybe bring the infection of unknown pathogens and spread of heterogen in the crowd. If cell resource is adequate, BAL and HCT will have a good prospect in clinical application. Recently hepatic stem cells has become a hot topic in the world. The hepatic stem cells are considered to be a kind of bipotential cells which can differentiate into hepatocytes and biliary epithelial cells. If we can proliferate hepatic stem cells and differentiate them into mature hepatocytes, we will solve above-mentioned problems about cell source. In this experiment we want to prove whether the human fetal HSCs can be induced and differentiated into hepatocytes and be used in the future. We will make some preparations for the clinical application of hepatic stem cells.Part I Isolation and culture of human fetal hepatic stem cells
Objective To establish a way of isolation, purification, identification and culture of human fetal hepatic stem cells. Methods Liver cells were isolated by collagenase (type IV) digestion method, and hepatic stem cells were purified from liver cells by Percoll discontinuous gradient centrifugat ion. Human fetal HSCs were obtained from the liver of aborted fetus in the second trimester of pregnancy. Under the asepsis conditions, establish umbilical-inferior vena extrinsic circulation. Perfuse EGTA balance liquid and collegen solution one by one. Then filtrate liver tissue by filter net and gain liver cells suspension. Centrifugate the liver cells suspension in three kinds of Percoll work solutions with different densities(1. llOg/ml, 1. 070g/ml, 1. 035g/ml). Extract the cells (HSCs) between the middle layer of Percoll and the bottom one. Then adjust the density of cells and culture them in DMEM/F12 media.The viability of hepatic stem cells was checked by trypan blue excluded test. The morphologic characters of human HSCs were observed by optic and electronic microscopy. The surface markers of HSCs including albumin, CK19, AFP and CD34 were identified by immuno- cytochemical staining. Count the amount of the cells during the whole process. Compute the yield of liver cells and human HSCs respectively. Results After two-step collagenase purfusion and centrifugation in discontinuous dentity Percoll solution, the HSCs could be purified from liver cells. The mean viability of HSCs is (88.35 + 2.45)% by trypan blue excluded test. By optic microscopy we can observe that HSCs have oval appearance and are smaller than mature hepatocytes. By electronic microscopy we can find that the diameter of HSCs is about 10 in, there are some microvilli on their surface, the oval nucleus occupy the most
space of the cell and there are few cytoplasm, endoplasmic reticula, mitochondria and other organelle. Immunocytochemical staining shows that the HSCs present the positive signal of CK19, CD34 and AFP and the negative signal of ALB. During short-term culture course the HSCs gradually adhere and begin to grow like epithelia. The mean yield of liver cells is (8. 72 + 1. 02) XlO'/liver (n = 15) , the mean yield of HSCs is (2.34+0.93) XlOVliver (n=15) or (8. 76+0. 46) X105yh/g (n=15) .Conclusions Human HSCs can be obtained by purfusion of collagenase solution and centrifugation of discontinuous dentity Percoll solution satisfactorily. We can acquire enough HSCs with considerable viability and use them for the following experiment.Part II Inducement and differentiation of human fetal hepaticstem cells with HGF and EGF in conditional culture mediaObjective Induce and differentiate human fetal HSCs into hepatocytes with HGF and EGF in conditional culture media. Methods Culture the HSCs in DMEM/F12 media with appropriate cell density as soon as they have been isolated. By MTT colorimetry observe the dose-effect relation of HGF or EGF in five groups which have different concentration of HGF or EGF except for one control group. In inducement experiment we devicle four groups, namely, negative control group, HGF group, EGF group and HGF+EGF group. Every three days replace the media and collect the specimens to be measured. Observe cells by optic microscopy and depict the growth-curves. Measure the content of urea
引文
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2. Faris RA, Konkin T, Halpert G. Liver stem cells: a potential sourse of hepatocytes for the treatment of human liver disease. Artificial Organs, 2001,25(7): 513-521.
3. Strain AJ, Crosby HA. Hepatic stem cells. Gut, 2000,46(6): 734-744.
4. Thorgeirsson SS. Hepatic stem cells in liver regeneration. FASEB Journal, 1996, 10(11): 1249-1256.
5. Alison MR, Poulsom R, Forbes S. Update on hepatic stem cells. Liver, 2001, 21(6): 367-373.
6. Vessey CJ, dela Hall PM. Hepatic stem cells:a review. Pathology, 2001,33(1),130-141.
7. Pertersen BE. Hepatic "stem" cells: coming full circle. Blood Cells Mol Dis, 2001, 27(3): 590-600.
8. Alison M, Sarraf C. Hepatic stem cells. Hepatology, 1998,29: 676-682.
9. Alison M. Liver stem cells: a two compartment system. Curr Opin Cell Biol,1998,10(6): 710-715.
10. Sell S. Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology, 2000, 31: 235-240.
11. Susick R, Moss N, Kubota H, et al. Hepatic progenitors and strateries for liver cell therapies. Annals of the New York Academy of Sciences, 2001, 944: 398-419.
12. Suzuki A, Nakauchi H, Taniguchi H, et al. In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells. Cell Transplant, 2003, 12(5): 469-473.
13. Alison M, Golding M, Lalani EN, et al. Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells. Hepatology, 1997, 26: 343-352.
14. Alison M, Golding M, Sarraf CE, et al. Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells. Gastroenter-ology, 1996, 110: 1182-1190.
15.张金卷,杜智,李涛等。大鼠肝卵原细胞的诱导、分离及鉴定。生物医学工程与临床,2004,8(4):196-199。
16. Dabeva MD, Petkov PM, Sandhu J, et al. Proliferation and differentia-tion of fetal liver epithelial progenitor cells after transplant-ation into adult rat liver. Pathology, 2000, 156: 2017-2031.
17. Sigal SH, Brill S, Reid LM.Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption and fluorescence-activated cell sorting.Hepatology, 1994,19:999-1006.
18. Monga SP, Tang Y,Candotti F, et al. Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants. Cell Transplant, 2001, 10(1):81-89.
19. Malhi H, Irani AN, Gagandeep S, et al. Isolation of human progenitor live epithelium cells with extensive replication capacity and differentiation into mature hepatocytes. Cell Science, 2002,115:2679-2688.
20. Haruna Y,Saito K, Spaulding S, et al.Identification of bipotential progenitor cells in human liver development. Hepatology, 1996, 23:476-481.
21. Seglen PO. Isolation of hepatocytes by collagenase perfusion. Methods Toxicology, 1993,1:231-234.
22. Gordon GJ, Butz GM, Grisham JW, et al. Isolation,short-term culture and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats. Transplantation, 2002, 73(8): 1236-1243.
23. Matsusaka S, Toyosaka A, Nakasho K, et al.The role of oval cells in rats hepatocyte transplantation. Transplantation, 2002, 70(3): 441-446.
24. Suzuki A, Zheng YW, Kondo R, et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology, 2002, 32(6):1230-1239.
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