内皮祖细胞和肝干细胞联合移植对大鼠肝纤维化的逆转作用
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摘要
背景:目前骨髓源性内皮祖细胞(bone marrow-derived endothelial progenitor cells,BM-EPCs)或骨髓源性肝干细胞(bone marrow-derived hepatocyte stem cells,BDHSCs)移植治疗肝纤维化的研究较多见,但二者联合移植治疗肝纤维化的报道却罕见。因此设想将具有血管形成作用的BM-EPCs和具有肝细胞再生作用的BDHSCs联合移植,可发挥双重抗纤维化作用。目的:探讨BM-EPCs和BDHSCs联合移植对大鼠肝纤维化的逆转作用。方法:利用四氯化碳皮下注射6周建立大鼠肝纤维化模型。通过体外诱导培养获得肝纤维化大鼠来源的BM-EPCs,通过磁珠分选获得肝纤维化大鼠来源的BDHSCs。将两种细胞经尾静脉、门静脉分支输注移植到肝纤维化大鼠体内,观察其对肝纤维化程度和肝脏功能的改善作用。结果与结论:①Masson染色结果显示,单纯BDHSCs或BM-EPCs移植,以及两者联合移植均能抑制胶原纤维的形成。但肝纤维化分期评分结果显示,仅BM-EPCs/BDHSCs联合移植能明显改善肝纤维化程度,与模型组相比差异有显著性意义(P<0.05);②肝功能和凝血功能检测结果显示,与模型组相比,BM-EPCs/BDHSCs联合移植组谷丙转氨酶、谷草转氨酶、总胆红素、凝血酶原时间和部分活化凝血活酶时间5项指标水平显著改善(P<0.05),与正常组水平相当;③结果表明,来源于肝纤维化大鼠的BM-EPCs/BDHSCs联合移植治疗肝纤维化有显著疗效,优于单一类型细胞移植。
BACKGROUND: At present, the transplantation of bone marrow-derived endothelial progenitor cells(BM-EPCs) or bone marrow-derived hepatocyte stem cells(BDHSCs) is common in the treatment of liver fibrosis, but the combined treatment for liver fibrosis is rarely reported. Combined transplantation of BM-EPCs possessing the function of angiogenesis and BDHSCs possessing the function of hepatocyte regeneration might play a dual anti-fibrosis role.OBJECTIVE: To evaluate the reversal effect on liver fibrosis by the combined transplantation of BM-EPCs and BDHSCs in rats. METHODS: The liver fibrosis rat models were induced with CCl4 subcutaneous injections for 6 weeks. BM-EPCs of rats with liver fibrosis were obtained by culture induction in vitro. BDHSCs of rats with liver fibrosis were obtained by magnetic bead cell sorting. BM-EPCs and/or BDHSCs were transplanted into liver fibrosis rats via the tail vein and branch of the portal vein, and then the effects of BDHSCs transplantatiron on liver fibrosis and liver function were observed. RESULTS AND CONCLUSION:(1) Masson staining results showed transplantations of BDHSCs and BM-EPCs, alone or both, could suppress the formation of collagen fibers. However, the staging scores of liver fibrosis showed that only the combined transplantation of BM-EPCs and BDHSCs could significantly improve liver fibrosis, which was significantly different from the model group(1.75±0.25 vs. 3.00±0.19, P < 0.05).(2) The liver biochemical assay in the blood showed that the levels of all five parameters of alanine aminotransferase, aspartate aminotransferase, total bilirubin, prothrombin time, and activated partial thromboplastin time in the BM-EPCs/BDHSCs group were significantly improved to be equivalent to normal levels, compared with those in the model group(P < 0.05). To conclude, it is an effective treatment for liver fibrosis by the co-transplantation of BM-EPCs and BDHSCs.
BACKGROUND: At present, the transplantation of bone marrow-derived endothelial progenitor cells(BM-EPCs) or bone marrow-derived hepatocyte stem cells(BDHSCs) is common in the treatment of liver fibrosis, but the combined treatment for liver fibrosis is rarely reported. Combined transplantation of BM-EPCs possessing the function of angiogenesis and BDHSCs possessing the function of hepatocyte regeneration might play a dual anti-fibrosis role.OBJECTIVE: To evaluate the reversal effect on liver fibrosis by the combined transplantation of BM-EPCs and BDHSCs in rats. METHODS: The liver fibrosis rat models were induced with CCl4 subcutaneous injections for 6 weeks. BM-EPCs of rats with liver fibrosis were obtained by culture induction in vitro. BDHSCs of rats with liver fibrosis were obtained by magnetic bead cell sorting. BM-EPCs and/or BDHSCs were transplanted into liver fibrosis rats via the tail vein and branch of the portal vein, and then the effects of BDHSCs transplantatiron on liver fibrosis and liver function were observed. RESULTS AND CONCLUSION:(1) Masson staining results showed transplantations of BDHSCs and BM-EPCs, alone or both, could suppress the formation of collagen fibers. However, the staging scores of liver fibrosis showed that only the combined transplantation of BM-EPCs and BDHSCs could significantly improve liver fibrosis, which was significantly different from the model group(1.75±0.25 vs. 3.00±0.19, P < 0.05).(2) The liver biochemical assay in the blood showed that the levels of all five parameters of alanine aminotransferase, aspartate aminotransferase, total bilirubin, prothrombin time, and activated partial thromboplastin time in the BM-EPCs/BDHSCs group were significantly improved to be equivalent to normal levels, compared with those in the model group(P < 0.05). To conclude, it is an effective treatment for liver fibrosis by the co-transplantation of BM-EPCs and BDHSCs.
引文
[1]Rautou PE.Endothelial progenitor cells in cirrhosis:the more,the merrier.J Hepatol.2012;57(6):1163-1165.
[2]Friedman SL.Mechanisms of hepatic fibrogenesis.Gastroenterology.2008;134(6):1655-1669.
[3]García-Bravo M,Morán-Jiménez MJ,Quintana-Bustamante O,et al.Bone marrow-derived cells promote liver regeneration in mice with erythropoietic protoporphyria.Transplantation.2009;88(12):1332-1340.
[4]Shackel N,Rockey D.In pursuit of the"Holy Grail"--stem cells,hepatic injury,fibrogenesis and repair.Hepatology.2005;41(1):16-18.
[5]Russo FP,Alison MR,Bigger BW,et al.The bone marrow functionally contributes to liver fibrosis.Gastroenterology.2006;130(6):1807-1821.
[6]Nussler A,Konig S,Ott M,et al.Present status and perspectives of cell-based therapies for liver diseases.J Hepatol.2006;45(1):144-159.
[7]Oyagi S,Hirose M,Kojima M,et al.Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats.J Hepatol.2006;44(4):742-748.
[8]Sakaida I,Terai S,Yamamoto N,et al.Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology.2004;40(6):1304-1311.
[9]Forbes SJ,Gupta S,Dhawan A.Cell therapy for liver disease:From liver transplantation to cell factory.J Hepatol.2015;62(1Suppl):S157-169.
[10]Ishikawa T,Terai S,Urata Y,et al.Fibroblast growth factor 2facilitates the differentiation of transplanted bone marrow cells into hepatocytes.Cell Tissue Res.2006;323(2):221-231.
[11]Lan L,Chen Y,Sun C,et al.Transplantation of bone marrow-derived hepatocyte stem cells transduced with adenovirus-mediated IL-10 gene reverses liver fibrosis in rats.Transpl Int.2008;21(6):581-592.
[12]Khoo CP,Pozzilli P,Alison MR.Endothelial progenitor cells and their potential therapeutic applications.Regen Med.2008;3(6):863-876.
[13]Sakamoto M,Nakamura T,Torimura T,et al.Transplantation of endothelial progenitor cells ameliorates vascular dysfunction and portal hypertension in carbon tetrachloride-induced rat liver cirrhotic model.J Gastroenterol Hepatol.2013;28(1):168-178.
[14]Nakamura T,Torimura T,Sakamoto M,et al.Significance and therapeutic potential of endothelial progenitor cell transplantation in a cirrhotic liver rat model.Gastroenterology.2007;133(1):91-107.
[15]Lian J,Lu Y,Xu P,et al.Prevention of liver fibrosis by intrasplenic injection of high-density cultured bone marrow cells in a rat chronic liver injury model.PLo S One.2014;9(9):e103603.
[16]Nakamura T,Torimura T,Iwamoto H,et al.Prevention of liver fibrosis and liver reconstitution of DMN-treated rat liver by transplanted EPCs.Eur J Clin Invest.2012;42(7):717-728.
[17]Taniguchi E,Kin M,Torimura T,et al.Endothelial progenitor cell transplantation improves the survival following liver injury in mice.Gastroenterology.2006;130(2):521-531.
[18]Liu F,Liu ZD,Wu N,et al.Transplanted endothelial progenitor cells ameliorate carbon tetrachloride-induced liver cirrhosis in rats.Liver Transpl.2009;15(9):1092-1100.
[19]周贤,刘翼,夏国栋,等.肝纤维化动物模型探讨[J].四川动物,2010,29(1):114-119.
[20]刘冉,兰玲,于静,等.肝纤维化大鼠骨髓源性内皮祖细胞的培养及鉴定[J].基础医学与临床杂志,2011,31(12):1335-1340.
[21]Avital I,Inderbitzin D,Aoki T,et al.Isolation,characterization,and transplantation of bone marrow-derived hepatocyte stem cells.Biochem Biophys Res Commun.2001;288(1):156-164.
[22]Inderbitzin D,Avital I,Gloor B,et al.Functional comparison of bone marrow-derived liver stem cells:selection strategy for cell-based therapy.J Gastrointest Surg.2005;9(9):1340-1345.
[23]Wu XL,Zeng WZ,Wang PL,et al.Effect of compound rhodiola sachalinensis A Bor on CCl4-induced liver fibrosis in rats and its probable molecular mechanisms.World J Gastroenterol.2003;9(7):1559-1562.
[24]兰玲,孙超,陈源文,等.大鼠β2m-/Thy-1+骨髓源性肝干细胞的体外分选及鉴定[J].上海交通大学学报:医学版,2007,27(11):1293-1296.
[25]姚鹏,胡大荣,王帅,等.自体骨髓干细胞移植治疗慢性重症肝病临床研究[J].透析与人工器官,2006,17(1):18-21.
[26]Thabut D,Shah V.Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease:new targets for the treatment of portal hypertension.J Hepatol.2010;53(5):976-980.
[27]Ueno T,Nakamura T,Torimura T,et al.Angiogenic cell therapy for hepatic fibrosis.Med Mol Morphol.2006;39(1):16-21.
[28]Fadini GP,Losordo D,Dimmeler S.Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use.Circ Res.2012;110(4):624-637.
[29]Casamassimi A,Balestrieri ML,Fiorito C,et al.Comparison between total endothelial progenitor cell isolation versus enriched Cd133+culture.J Biochem.2007;141(4):503-511.
[30]曹葆强,林继宗,钟跃思,等.自体骨髓细胞经门静脉移植治疗肝硬化与肝功能不全的临床研究[J].中华普通外科杂志,2007,22(5):386-389.
[31]Kushida T,Inaba M,Hisha H,et al.Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein.Stem Cells.2001;19(3):226-235.
[32]兰玲,于静,刘冉,等.骨髓内皮祖细胞输注后在肝纤维化大鼠肝脏内的示踪研究[J].中华器官移植杂志,2012,33(11):684-688.
[33]刘冉,兰玲,刘博伟,等.来源于肝纤维化环境内皮祖细胞移植治疗肝纤维化模型大鼠的逆转作用[J].中国组织工程研究,2017,21(13):2068-2073.
[34]王喜军,孙文军,孙晖,等.CCL4诱导大鼠肝损伤模型的代谢组学及茵陈蒿汤的干预作用研究[J].世界科学技术-中医药现代化,2006,8(6):101-106.
[35]万智,安宁,朱易萍,等.丙氨酸转氨酶的研究现状与进展[J].华西医学,2010,25(1):238-240.
[36]刁建新,马文校,刘亚伟,等.大鼠急性肝功能衰竭模型的建立及凝血功能的变化[J].世界科学技术-中医药现代化,2014,16(11):2406-2410.
[2]Friedman SL.Mechanisms of hepatic fibrogenesis.Gastroenterology.2008;134(6):1655-1669.
[3]García-Bravo M,Morán-Jiménez MJ,Quintana-Bustamante O,et al.Bone marrow-derived cells promote liver regeneration in mice with erythropoietic protoporphyria.Transplantation.2009;88(12):1332-1340.
[4]Shackel N,Rockey D.In pursuit of the"Holy Grail"--stem cells,hepatic injury,fibrogenesis and repair.Hepatology.2005;41(1):16-18.
[5]Russo FP,Alison MR,Bigger BW,et al.The bone marrow functionally contributes to liver fibrosis.Gastroenterology.2006;130(6):1807-1821.
[6]Nussler A,Konig S,Ott M,et al.Present status and perspectives of cell-based therapies for liver diseases.J Hepatol.2006;45(1):144-159.
[7]Oyagi S,Hirose M,Kojima M,et al.Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats.J Hepatol.2006;44(4):742-748.
[8]Sakaida I,Terai S,Yamamoto N,et al.Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology.2004;40(6):1304-1311.
[9]Forbes SJ,Gupta S,Dhawan A.Cell therapy for liver disease:From liver transplantation to cell factory.J Hepatol.2015;62(1Suppl):S157-169.
[10]Ishikawa T,Terai S,Urata Y,et al.Fibroblast growth factor 2facilitates the differentiation of transplanted bone marrow cells into hepatocytes.Cell Tissue Res.2006;323(2):221-231.
[11]Lan L,Chen Y,Sun C,et al.Transplantation of bone marrow-derived hepatocyte stem cells transduced with adenovirus-mediated IL-10 gene reverses liver fibrosis in rats.Transpl Int.2008;21(6):581-592.
[12]Khoo CP,Pozzilli P,Alison MR.Endothelial progenitor cells and their potential therapeutic applications.Regen Med.2008;3(6):863-876.
[13]Sakamoto M,Nakamura T,Torimura T,et al.Transplantation of endothelial progenitor cells ameliorates vascular dysfunction and portal hypertension in carbon tetrachloride-induced rat liver cirrhotic model.J Gastroenterol Hepatol.2013;28(1):168-178.
[14]Nakamura T,Torimura T,Sakamoto M,et al.Significance and therapeutic potential of endothelial progenitor cell transplantation in a cirrhotic liver rat model.Gastroenterology.2007;133(1):91-107.
[15]Lian J,Lu Y,Xu P,et al.Prevention of liver fibrosis by intrasplenic injection of high-density cultured bone marrow cells in a rat chronic liver injury model.PLo S One.2014;9(9):e103603.
[16]Nakamura T,Torimura T,Iwamoto H,et al.Prevention of liver fibrosis and liver reconstitution of DMN-treated rat liver by transplanted EPCs.Eur J Clin Invest.2012;42(7):717-728.
[17]Taniguchi E,Kin M,Torimura T,et al.Endothelial progenitor cell transplantation improves the survival following liver injury in mice.Gastroenterology.2006;130(2):521-531.
[18]Liu F,Liu ZD,Wu N,et al.Transplanted endothelial progenitor cells ameliorate carbon tetrachloride-induced liver cirrhosis in rats.Liver Transpl.2009;15(9):1092-1100.
[19]周贤,刘翼,夏国栋,等.肝纤维化动物模型探讨[J].四川动物,2010,29(1):114-119.
[20]刘冉,兰玲,于静,等.肝纤维化大鼠骨髓源性内皮祖细胞的培养及鉴定[J].基础医学与临床杂志,2011,31(12):1335-1340.
[21]Avital I,Inderbitzin D,Aoki T,et al.Isolation,characterization,and transplantation of bone marrow-derived hepatocyte stem cells.Biochem Biophys Res Commun.2001;288(1):156-164.
[22]Inderbitzin D,Avital I,Gloor B,et al.Functional comparison of bone marrow-derived liver stem cells:selection strategy for cell-based therapy.J Gastrointest Surg.2005;9(9):1340-1345.
[23]Wu XL,Zeng WZ,Wang PL,et al.Effect of compound rhodiola sachalinensis A Bor on CCl4-induced liver fibrosis in rats and its probable molecular mechanisms.World J Gastroenterol.2003;9(7):1559-1562.
[24]兰玲,孙超,陈源文,等.大鼠β2m-/Thy-1+骨髓源性肝干细胞的体外分选及鉴定[J].上海交通大学学报:医学版,2007,27(11):1293-1296.
[25]姚鹏,胡大荣,王帅,等.自体骨髓干细胞移植治疗慢性重症肝病临床研究[J].透析与人工器官,2006,17(1):18-21.
[26]Thabut D,Shah V.Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease:new targets for the treatment of portal hypertension.J Hepatol.2010;53(5):976-980.
[27]Ueno T,Nakamura T,Torimura T,et al.Angiogenic cell therapy for hepatic fibrosis.Med Mol Morphol.2006;39(1):16-21.
[28]Fadini GP,Losordo D,Dimmeler S.Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use.Circ Res.2012;110(4):624-637.
[29]Casamassimi A,Balestrieri ML,Fiorito C,et al.Comparison between total endothelial progenitor cell isolation versus enriched Cd133+culture.J Biochem.2007;141(4):503-511.
[30]曹葆强,林继宗,钟跃思,等.自体骨髓细胞经门静脉移植治疗肝硬化与肝功能不全的临床研究[J].中华普通外科杂志,2007,22(5):386-389.
[31]Kushida T,Inaba M,Hisha H,et al.Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein.Stem Cells.2001;19(3):226-235.
[32]兰玲,于静,刘冉,等.骨髓内皮祖细胞输注后在肝纤维化大鼠肝脏内的示踪研究[J].中华器官移植杂志,2012,33(11):684-688.
[33]刘冉,兰玲,刘博伟,等.来源于肝纤维化环境内皮祖细胞移植治疗肝纤维化模型大鼠的逆转作用[J].中国组织工程研究,2017,21(13):2068-2073.
[34]王喜军,孙文军,孙晖,等.CCL4诱导大鼠肝损伤模型的代谢组学及茵陈蒿汤的干预作用研究[J].世界科学技术-中医药现代化,2006,8(6):101-106.
[35]万智,安宁,朱易萍,等.丙氨酸转氨酶的研究现状与进展[J].华西医学,2010,25(1):238-240.
[36]刁建新,马文校,刘亚伟,等.大鼠急性肝功能衰竭模型的建立及凝血功能的变化[J].世界科学技术-中医药现代化,2014,16(11):2406-2410.