自体来源诱导性多能干细胞移植治疗小鼠慢性乙型肝炎性肝损伤
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摘要
背景:自体来源诱导性多能干细胞移植改善肝组织结构与功能是慢性乙型肝炎性肝损伤治疗的新研究方向。目的:观察自体皮肤来源诱导性多能干细胞移植治疗HBV转基因小鼠慢性肝损伤的效果。方法:用腹腔注射四氯化碳建立HBV转基因小鼠(购于北京维通达生物技术有限公司)慢性肝损伤模型;造模开始注射四氯化碳第1周后,将模型小鼠皮肤成纤维细胞体外重编程为诱导性多能干细胞并进行鉴定;造模6周结束后第2天,通过门静脉途径将0.1 mL诱导性多能干细胞移植到模型小鼠肝内,对照组注射0.1 mL PBS;移植后7,14 d,冰冻切片观察诱导性多能干细胞定植情况;ELISA法检测小鼠肝功能;石蜡切片苏木精-伊红染色观察肝组织病理学变化。结果与结论:(1)模型小鼠皮肤成纤维细胞在体外成功重编程为诱导性多能干细胞,具有多向分化潜能;(2)移植后7 d,实验组小鼠肝内可见荧光标记的诱导性多能干细胞;(3)实验组小鼠血清谷丙转氨酶、谷草转氨酶、谷氨酰转肽酶、总胆红素水平低于对照组,白蛋白水平高于对照组(P <0.05);(4)实验组小鼠肝脏中肝细胞变性、坏死及炎细胞浸润较对照组明显改善;(5)结果提示,自体来源诱导性多能干细胞门静脉移植可治疗小鼠慢性乙型肝炎肝损伤。
BACKGROUND:Autologous source induced pluripotent stem cell transplantation to improve liver tissue structure and function is a new research direction in the treatment of liver injury caused by chronic hepatitis B.OBJECTIVE:To observe the effect of autologous skin-derived induced pluripotent stem cell transplantation on chronic hepatic injury in HBV transgenic mice.METHODS:A chronic hepatic injury model of HBV transgenic mice(provided by the Beijing Vitalstar Biotechnology Co., Ltd. in China) was established with intraperitoneal injection of carbon tetrachloride. At 1 week after injection of carbon tetrachloride, skin fibroblasts from the model mice were reprogrammed into induced pluripotent stem cells and identified in vitro. Induced pluripotent stem cells(0.1 mL) were transplanted into the liver of model mice through the portal vein pathway as experimental group, and PBS(0.1 mL) was injected into the other mice as control group. Colonization of induced pluripotent stem cells was observed by frozen section at 7 and 14 days of transplantation. Liver function of the mice was detected by ELISA, and liver histopathological changes were observed by hematoxylin-eosin staining of paraffin section.RESULTS AND CONCLUSION:Skin fibroblasts from model mice were successfully reprogrammed into induced pluripotent stem cells in vitro that had multipotent differentiation potential. At 7 days after transplantation, fluorescent labeled induced pluripotent stem cells were found in the mouse liver of the experimental group. The levels of serum alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase, and total bilirubin in the experimental group were lower than those in the control group, and the level of albumin was higher than that in the control group(P < 0.05). Liver cell degeneration, necrosis and inflammatory cell infiltration in the mouse liver of the experimental group were significantly improved as compared with the control group. These results suggest that portal vein transplantation of autologous source induced pluripotent stem cells can treat chronic hepatitis B-induced liver injury in mice.
BACKGROUND:Autologous source induced pluripotent stem cell transplantation to improve liver tissue structure and function is a new research direction in the treatment of liver injury caused by chronic hepatitis B.OBJECTIVE:To observe the effect of autologous skin-derived induced pluripotent stem cell transplantation on chronic hepatic injury in HBV transgenic mice.METHODS:A chronic hepatic injury model of HBV transgenic mice(provided by the Beijing Vitalstar Biotechnology Co., Ltd. in China) was established with intraperitoneal injection of carbon tetrachloride. At 1 week after injection of carbon tetrachloride, skin fibroblasts from the model mice were reprogrammed into induced pluripotent stem cells and identified in vitro. Induced pluripotent stem cells(0.1 mL) were transplanted into the liver of model mice through the portal vein pathway as experimental group, and PBS(0.1 mL) was injected into the other mice as control group. Colonization of induced pluripotent stem cells was observed by frozen section at 7 and 14 days of transplantation. Liver function of the mice was detected by ELISA, and liver histopathological changes were observed by hematoxylin-eosin staining of paraffin section.RESULTS AND CONCLUSION:Skin fibroblasts from model mice were successfully reprogrammed into induced pluripotent stem cells in vitro that had multipotent differentiation potential. At 7 days after transplantation, fluorescent labeled induced pluripotent stem cells were found in the mouse liver of the experimental group. The levels of serum alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase, and total bilirubin in the experimental group were lower than those in the control group, and the level of albumin was higher than that in the control group(P < 0.05). Liver cell degeneration, necrosis and inflammatory cell infiltration in the mouse liver of the experimental group were significantly improved as compared with the control group. These results suggest that portal vein transplantation of autologous source induced pluripotent stem cells can treat chronic hepatitis B-induced liver injury in mice.
引文
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[8]丛姗,白立恒,李岩,等.人羊膜间充质干细胞移植对CCl4诱导的小鼠损伤肝HGF、SIRT-1、α-SMA及P~(27kip1)表达的影响[J].中国生物化学与分子生物学报,2015,31(3):292-300.
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[10]吴玉卓,杨乐,翟玉峰,等.自体骨髓间充质干细胞治疗乙型肝炎失代偿期患者:肝纤维化、肝功能、MELD评分及1年生存率分析[J].中国组织工程研究,2017,21(13):2049-2055.
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[12]Pocock JM,Piers TM.Modelling microglial function with induced pluripotent stem cells:an update.Nat Rev Neurosci.2018;19(8):445-452.
[13]张雪,明雅南,张岚.诱导性多能干细胞的安全性及其在肝细胞再生方面的应用潜能[J].肝脏,2016,21(5):404-407.
[14]Roy-Chowdhury N,Wang X,Guha C,et al.Hepatocyte-like cells derived from induced pluripotent stem cells.Hepatol Int.2017;11(1):54-69.
[15]Pournasr B,Duncan SA.Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.Arterioscler Thromb Vasc Biol.2017;37(11):1994-1999.
[16]李君,杜潇,吴嫣爽,等.不同分化阶段诱导性多能干细胞移植实验研究进展[J].解剖学报,2015,46(5):699-703.
[17]Revill P,Yuan Z.New insights into how HBV manipulates the innate immune response to establish acute and persistent infection.Antivir Ther.2013;18(1):1-15.
[18]Chang RH,Nyamsuren T,Gyawali S,et al.Long-term Nucleos(t)ides Analogues for Chronic Hepatitis B Improve Liver and Spleen Size:A Noninvasive Sonographic Study.J Med Ultrasound.2017;25(3):161-166.
[19]Raven S,Urbanus A,de Gee A,et al.Predictors of hepatitis Bvaccination completion among people who use drugs participating in a national program of targeted vaccination.Vaccine.2018;36(35):5282-5287.
[20]Liu D,Liu L,Wang L,et al.Therapeutic effects of mesenchymal stem cells combined with short hairpin RNA on liver injury induced by hepatitis B virus infection.Mol Med Rep.2018;17(1):1731-1741.
[21]Berenguer M,de la Rosa Rodríguez G,Domínguez-Gil B.Significant impact of new oral therapies against HCV on the waiting list for liver transplantation in Spain.J Hepatol.2018;69(4):966-968.
[22]Zheng WP,Zhang BY,Shen ZY,et al.Biological effects of bone marrow mesenchymal stem cells on hepatitis B virus in vitro.Mol Med Rep.2017;15(5):2551-2559.
[23]Qu M,Yuan X,Liu D,et al.Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Immune-Mediated Liver Injury and Compromise Virus Control During Acute Hepatitis B Virus Infection in Mice.Stem Cells Dev.2017;26(11):818-827.
[24]Lou C,Bai T,Bi LW,et al.Negative impact of hepatitis B surface seroclearance on prognosis of hepatitis B-related primary liver cancer.World J Clin Cases.2018;6(8):192-199.
[25]Tseng CH,Chang CY,Mo LR,et al.Acoustic Radiation Force Impulse Elastography with APRI and FIB-4 to Identify Significant Liver Fibrosis in Chronic Hepatitis B Patients.Ann Hepatol.2018;17(5):789-794.
[26]Singh K,Evens H,Nair N,et al.Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9.Mol Ther.2018;26(5):1241-1254.
[27]Aravalli RN,Steer CJ.Gene editing technology as an approach to the treatment of liver diseases.Expert Opin Biol Ther.2016;16(5):595-608.
[28]Sukowati CH,El-Khobar KE,Ie SI,et al.Significance of hepatitis virus infection in the oncogenic initiation of hepatocellular carcinoma.World J Gastroenterol.2016;22(4):1497-1512.
[29]Terai S,Takami T,Yamamoto N,et al.Status and prospects of liver cirrhosis treatment by using bone marrow-derived cells and mesenchymal cells.Tissue Eng Part B Rev.2014;20(3):206-210.
[30]Li YH,Xu Y,Wu HM,et al.Umbilical Cord-Derived Mesenchymal Stem Cell Transplantation in Hepatitis B Virus Related Acute-onChronic Liver Failure Treated with Plasma Exchange and Entecavir:a 24-Month Prospective Study.Stem Cell Rev.2016;12(6):645-653.
[2]Bhatia M,Gupta E,Choudhary MC,et al.Evaluation of impact of occult hepatitis B infection in chronic HCV-infected patients:Aretrospective cohort study.J Lab Physicians.2018;10(3):304-308.
[3]Lanini S,Pisapia R,Capobianchi MR,et al.Global epidemiology of viral hepatitis and national needs for complete control.Expert Rev Anti Infect Ther.2018;16(8):625-639.
[4]Jiang XW,Ye JZ,Li YT,et al.Hepatitis B reactivation in patients receiving direct-acting antiviral therapy or interferon-based therapy for hepatitis C:A systematic review and meta-analysis.World JGastroenterol.2018;24(28):3181-3191.
[5]闫成,薛改,吴丽颖,等.两种肝组织匀浆上清液诱导人脐带间充质干细胞向肝细胞分化的比较[J].中国组织工程研究,2015,19(19):2993-2998.
[6]袁淑芳,胡兰英,姜涛,等.骨髓间充质干细胞移植肝衰竭大鼠CD163和白细胞介素10的表达[J].中国组织工程研究,2014,18(6):919-925.
[7]赵文秀,张磊,刘建明,等.小鼠脂肪间充质干细胞的分离培养及其在肝内归巢[J].南方医科大学学报,2013,33(8):1151-1154.
[8]丛姗,白立恒,李岩,等.人羊膜间充质干细胞移植对CCl4诱导的小鼠损伤肝HGF、SIRT-1、α-SMA及P~(27kip1)表达的影响[J].中国生物化学与分子生物学报,2015,31(3):292-300.
[9]王建军,赵平,靳雪源,等.诱导性多能干细胞治疗小鼠肝损伤的初步研究[J].中国肝脏病杂志(电子版),2016,8(2):39-43.
[10]吴玉卓,杨乐,翟玉峰,等.自体骨髓间充质干细胞治疗乙型肝炎失代偿期患者:肝纤维化、肝功能、MELD评分及1年生存率分析[J].中国组织工程研究,2017,21(13):2049-2055.
[11]任浩,董静,刘力伟,等.人脐带间充质干细胞治疗乙型肝炎肝硬化患者发生肝细胞癌的危险因素分析[J].中国组织工程研究,2018,22(9):1350-1356.
[12]Pocock JM,Piers TM.Modelling microglial function with induced pluripotent stem cells:an update.Nat Rev Neurosci.2018;19(8):445-452.
[13]张雪,明雅南,张岚.诱导性多能干细胞的安全性及其在肝细胞再生方面的应用潜能[J].肝脏,2016,21(5):404-407.
[14]Roy-Chowdhury N,Wang X,Guha C,et al.Hepatocyte-like cells derived from induced pluripotent stem cells.Hepatol Int.2017;11(1):54-69.
[15]Pournasr B,Duncan SA.Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.Arterioscler Thromb Vasc Biol.2017;37(11):1994-1999.
[16]李君,杜潇,吴嫣爽,等.不同分化阶段诱导性多能干细胞移植实验研究进展[J].解剖学报,2015,46(5):699-703.
[17]Revill P,Yuan Z.New insights into how HBV manipulates the innate immune response to establish acute and persistent infection.Antivir Ther.2013;18(1):1-15.
[18]Chang RH,Nyamsuren T,Gyawali S,et al.Long-term Nucleos(t)ides Analogues for Chronic Hepatitis B Improve Liver and Spleen Size:A Noninvasive Sonographic Study.J Med Ultrasound.2017;25(3):161-166.
[19]Raven S,Urbanus A,de Gee A,et al.Predictors of hepatitis Bvaccination completion among people who use drugs participating in a national program of targeted vaccination.Vaccine.2018;36(35):5282-5287.
[20]Liu D,Liu L,Wang L,et al.Therapeutic effects of mesenchymal stem cells combined with short hairpin RNA on liver injury induced by hepatitis B virus infection.Mol Med Rep.2018;17(1):1731-1741.
[21]Berenguer M,de la Rosa Rodríguez G,Domínguez-Gil B.Significant impact of new oral therapies against HCV on the waiting list for liver transplantation in Spain.J Hepatol.2018;69(4):966-968.
[22]Zheng WP,Zhang BY,Shen ZY,et al.Biological effects of bone marrow mesenchymal stem cells on hepatitis B virus in vitro.Mol Med Rep.2017;15(5):2551-2559.
[23]Qu M,Yuan X,Liu D,et al.Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Immune-Mediated Liver Injury and Compromise Virus Control During Acute Hepatitis B Virus Infection in Mice.Stem Cells Dev.2017;26(11):818-827.
[24]Lou C,Bai T,Bi LW,et al.Negative impact of hepatitis B surface seroclearance on prognosis of hepatitis B-related primary liver cancer.World J Clin Cases.2018;6(8):192-199.
[25]Tseng CH,Chang CY,Mo LR,et al.Acoustic Radiation Force Impulse Elastography with APRI and FIB-4 to Identify Significant Liver Fibrosis in Chronic Hepatitis B Patients.Ann Hepatol.2018;17(5):789-794.
[26]Singh K,Evens H,Nair N,et al.Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9.Mol Ther.2018;26(5):1241-1254.
[27]Aravalli RN,Steer CJ.Gene editing technology as an approach to the treatment of liver diseases.Expert Opin Biol Ther.2016;16(5):595-608.
[28]Sukowati CH,El-Khobar KE,Ie SI,et al.Significance of hepatitis virus infection in the oncogenic initiation of hepatocellular carcinoma.World J Gastroenterol.2016;22(4):1497-1512.
[29]Terai S,Takami T,Yamamoto N,et al.Status and prospects of liver cirrhosis treatment by using bone marrow-derived cells and mesenchymal cells.Tissue Eng Part B Rev.2014;20(3):206-210.
[30]Li YH,Xu Y,Wu HM,et al.Umbilical Cord-Derived Mesenchymal Stem Cell Transplantation in Hepatitis B Virus Related Acute-onChronic Liver Failure Treated with Plasma Exchange and Entecavir:a 24-Month Prospective Study.Stem Cell Rev.2016;12(6):645-653.
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