急性肝衰竭模型猪微环境对脂肪间充质干细胞生物学特性的影响
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摘要
目的探究急性肝衰竭(ALF)猪脂肪来源的间充质干细胞(ADMSCs)的生物学活性,为ALF-ADMSCs是否具有潜在的自体移植潜能提供实验依据。方法分别获取急性肝衰竭猪来源的ADMSCs和正常猪来源的ADMSCs (Nor-ADMSCs),进行原代分离和培养。观察第1代与第5代ALF-ADMSCs的细胞形态,CCK8法检测ALF-ADMSCs增殖能力,检测和分析其诱导分化为成骨细胞、脂肪细胞、成软骨细胞、成肝细胞的能力,以及分析ALF-ADMSCs和Nor-ADMSCs中肝脏特异性基因的表达。结果成功分离培养ALF-ADMSCs和Nor-ADMSCs,第1代与第5代ALFADMSCs细胞形态均与Nor-ADMSCs相似。ALF-ADMSCs和Nor-ADMSCs均高表达间充质干细胞表面标记CD105和CD90,不表达或低表达造血干细胞表面标记CD14。ALF-ADMSCs与NorADMSCs增殖能力差异无统计学意义(P>0.05)。与Nor-ADMSCs相比较,ALF-ADMSCs诱导分化为成骨细胞、脂肪细胞、软骨细胞、肝样细胞的能力差异无统计学意义(P>0.05)。肝细胞生长因子(HGF)在ALF-ADMSCs中表达量比Nor-ADMSCs高(12.48±0.59)倍。结论 ALF-ADMSCs具有正常的干细胞特征与生物学特性,没有因ALF微环境而改变,且其高表达肝细胞生长因子,或可作为干细胞移植治疗终末期肝病的理想细胞源。
Objective To explore the biological characteristics of adipose-derived mesenchymal stem cells collected from pigs with acute liver failure(ALF-ADMSCs), in order to providing an experimental basis for the potential application of ADMSCs autologous transplantation in patients with end-stage liver disease. Methods ADMSCs isolated from acute liver failure pig and from a normal pig(Nor-ADMSCs) were primary cultured, respectively. The morphology of the first and fifth generation ALF-ADMSCs was observed. The cell proliferation of ALF-ADMSCs was detected by CCK8. Also, its ability to induce and differentiate into osteoblasts, adipocytes, chondrocytes and hepatoblasts were evaluated. The expression of liver-specific genes in ALF-ADMSCs and Nor-ADMSCs were analyzed. Results ALF-ADMSCs and Nor-ADMSCs were successfully isolated and cultured. The morphology of the first and fifth generation ALF-ADMSCs was similar to that of Nor-ADMSCs. Both ALF-ADMSCs and Nor-ADMSCs highly expressed the surface marker CD105 and CD90 of mesenchymal stem cells, but did not express or lowly expressed the surface marker CD14 of hematopoietic stem cells. There was no difference in proliferation ability between ALF-ADMSCs and Nor-ADMSCs. Compared to Nor-ADMSCs, ALF-ADMSCs have comparable ability to induce differentiation into osteoblasts, adipocytes, chondrocytes and hepatocyte-like cells. The expression levels of hepatocyte growth factor(HGF) in ALF-ADMSCs was 12.48±0.59 times higher than that of Nor-ADMSCs. Conclusion The results suggested that the stem cell features and biological characteristics of ALF-ADMSCs could remain under the acute liver failure microenvironment. ALF-ADMSCs highly expressed HGF, which indicated that it would be an ideal stem cell source for autologous transplantation in treating end-stage liver disease.
Objective To explore the biological characteristics of adipose-derived mesenchymal stem cells collected from pigs with acute liver failure(ALF-ADMSCs), in order to providing an experimental basis for the potential application of ADMSCs autologous transplantation in patients with end-stage liver disease. Methods ADMSCs isolated from acute liver failure pig and from a normal pig(Nor-ADMSCs) were primary cultured, respectively. The morphology of the first and fifth generation ALF-ADMSCs was observed. The cell proliferation of ALF-ADMSCs was detected by CCK8. Also, its ability to induce and differentiate into osteoblasts, adipocytes, chondrocytes and hepatoblasts were evaluated. The expression of liver-specific genes in ALF-ADMSCs and Nor-ADMSCs were analyzed. Results ALF-ADMSCs and Nor-ADMSCs were successfully isolated and cultured. The morphology of the first and fifth generation ALF-ADMSCs was similar to that of Nor-ADMSCs. Both ALF-ADMSCs and Nor-ADMSCs highly expressed the surface marker CD105 and CD90 of mesenchymal stem cells, but did not express or lowly expressed the surface marker CD14 of hematopoietic stem cells. There was no difference in proliferation ability between ALF-ADMSCs and Nor-ADMSCs. Compared to Nor-ADMSCs, ALF-ADMSCs have comparable ability to induce differentiation into osteoblasts, adipocytes, chondrocytes and hepatocyte-like cells. The expression levels of hepatocyte growth factor(HGF) in ALF-ADMSCs was 12.48±0.59 times higher than that of Nor-ADMSCs. Conclusion The results suggested that the stem cell features and biological characteristics of ALF-ADMSCs could remain under the acute liver failure microenvironment. ALF-ADMSCs highly expressed HGF, which indicated that it would be an ideal stem cell source for autologous transplantation in treating end-stage liver disease.
引文
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[15]Saidi R,Rajeshkumar R,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells promote liver regeneration[J].J Invest Surg,2015,28(6):303-308.
[16]Zhang J,Zhou S,Zhou Y,et al.Hepatocyte growth factor gene-modified adipose-derived mesenchymal stem cells ameliorate radiation induced liver damage in a rat model[J].PLoS One,2014,9(12):e114670.
[2]Gilsanz C,Aller MA,Fuentes-Julian S,et al.Adiposederived mesenchymal stem cells slow disease progression of acute-on-chronic liver failure[J].Biomed Pharmacother,2017,91():776-787.
[3]Zagoura DS,Roubelakis MG,Bitsika V,et al.Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure[J].Gut,2012,61(6):894-906.
[4]Jung J,Choi JH,Lee Y,et al.Human placenta-derived mesenchymal stem cells promote hepatic regeneration in CCl4-injured rat liver model via increased autophagic mechanism[J].Stem Cells,2013,31(8):1584-1596.
[5]Zhang Z,Lin H,Shi M,et al.Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients[J].J Gastroenterol Hepatol,2012,2():112-120.
[6]Wang Y,Wang F,Zhao H,et al.Human adipose-derived mesenchymal stem cells are resistant to HBV infection during differentiation into hepatocytes in vitro[J].Int J Mol Sci,2014,15(4):6096-6110.
[7]Mohsin S,Shams S,Ali Nasir G,et al.Enhanced hepatic differentiation of mesenchymal stem cells after pretreatment with injured liver tissue[J].Differentiation,2011,81(1):42-48.
[8]Yang Y,Qu B,Huo JH,et al.Serum from radiofrequencyinjured livers induces differentiation of bone marrow stem cells into hepatocyte-like cells[J].J.Surg.Res,2009,155(1):18-24.
[9]Moon GJ,Cho YH,Kim DH,et al.Serum-mediated activation of bone marrow-derived mesenchymal stem cells in ischemic stroke patients:a novel preconditioning method[J].Cell Transplant,2018,27(3):485-500.
[10]Shi D,Zhang J,Zhou Q,et al.Quantitative evaluation of human bone mesenchymal stem cells rescuing fulminant hepatic failure in pigs[J].Gut,2017,66(5):955-964.
[11]Zhang S,Zhu Z,Wang Y,et al.Therapeutic potential of Bama miniature pig adipose stem cells induced hepatocytes in a mouse model with acute liver failure[J].Cytotechnology,2018,70(4):1131-1141.
[12]Lee CW,Chen YF,Wu HH,et al.Historical perspectives and advances in mesenchymal stem cell research for the treatment of liver diseases[J].Gastroenterology,2018,154(1):46-56.
[13]Tsekouras A,Mantas D,Tsilimigras DI,et al.Comparison of the viability and yield of adipose-derived stem cells(ASCs)from different donor areas[J].In Vivo,2017,31(6):1229-1234.
[14]Teshima T,Matsumoto H,Michishita M,et al.Allogenic adipose tissue-derived mesenchymal stem cells ameliorate acute hepatic injury in dogs[J].Stem Cells Int,2017,2017:3892514.
[15]Saidi R,Rajeshkumar R,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells promote liver regeneration[J].J Invest Surg,2015,28(6):303-308.
[16]Zhang J,Zhou S,Zhou Y,et al.Hepatocyte growth factor gene-modified adipose-derived mesenchymal stem cells ameliorate radiation induced liver damage in a rat model[J].PLoS One,2014,9(12):e114670.