骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞
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摘要
目的探讨大鼠骨髓间充质干细胞(BMSCs)与肝星状细胞(HSCs)体外共培养体系中,BMSCs旁分泌肝细胞生长因子(HGF)对HSCs增殖、凋亡、活化的影响。方法全骨髓贴壁法分离、培养、纯化大鼠BMSCs,另培养HSCs。6孔板半透膜建立上下两层细胞非直接接触共培养体系,实验设H组(HSCs单独培养)、H-H组(HSCs与HSCs共培养)、M-H组(BMSCs与HSCs共培养)、M-H-C组(BMSCs与HSCs共培养并加c-met抑制剂),各组细胞培养48 h后,流式细胞仪鉴定BMSCs,检测HSCs凋亡率,MTT法检测HSCs的增殖,免疫荧光共聚焦定量检测HSCs中α-肌动蛋白(α-SMA)的表达量,ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子CD29、CD90,低表达造血细胞表面标记CD45;BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡,且M-H组上清液中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中,BMSCs通过旁分泌HGF促进HSCs的凋亡,抑制HSCs的增殖、活化。
Objective To investigate the effects of paracrine hepatocyte growth factor(HGF) secreted by rat bone marrow mesenchmal stem cells on proliferation, apoptosis and activation of hepatic stellate cells after rat bone marrow mesenhymal stem cells(BMSCs) and hepatic stellate cells(HSCs) were co-cultured in vitro. Methods The whole bone marrow adherent method was used to isolate, culture and purify rat BMSCs, and HSCs were cultured. The semi-permeable membrane of six-well plate was used to establish a two-layer cell co-culture system with indirect contact. The study consisted of group H(HSCs were cultured alone), H-H group(HSCs and HSCs were co-cultured), M-H group(BMSCs and HSCs were co-cultured), M-H-C group(BMSCs and HSCs were co-cultured with c-met inhibitor). Cells in each group were cultured for 48 hours. BMSCs were identified and the apoptosis rate of HSCs was detected by flow cytometry. The proliferation of HSCs was detected by MTT assay, and the expression level of alpha smooth muscle actin(α-SMA) in HSCs was detected quantitatively by immunofluorescence confocal method. The concentration of HGF in the supernate of the coculture system was determined by ELISA. Results MSCs overexpressed positive surface molecules CD29 and CD90, and low expression of hematopoietic cell surface marker CD45. BMSCs could significantly inhibit the proliferation and activation of HSCs, and promote apoptosis of HSCs. The concentration of HGF in the supernate was significantly higher in M-H group than that in the other groups. Conclusion BMSCs can promote the apoptosis of HSCs, inhibit the proliferation and activation of HSCs through paracrine HGF in the co-culture of BMSCs and HSCs.
Objective To investigate the effects of paracrine hepatocyte growth factor(HGF) secreted by rat bone marrow mesenchmal stem cells on proliferation, apoptosis and activation of hepatic stellate cells after rat bone marrow mesenhymal stem cells(BMSCs) and hepatic stellate cells(HSCs) were co-cultured in vitro. Methods The whole bone marrow adherent method was used to isolate, culture and purify rat BMSCs, and HSCs were cultured. The semi-permeable membrane of six-well plate was used to establish a two-layer cell co-culture system with indirect contact. The study consisted of group H(HSCs were cultured alone), H-H group(HSCs and HSCs were co-cultured), M-H group(BMSCs and HSCs were co-cultured), M-H-C group(BMSCs and HSCs were co-cultured with c-met inhibitor). Cells in each group were cultured for 48 hours. BMSCs were identified and the apoptosis rate of HSCs was detected by flow cytometry. The proliferation of HSCs was detected by MTT assay, and the expression level of alpha smooth muscle actin(α-SMA) in HSCs was detected quantitatively by immunofluorescence confocal method. The concentration of HGF in the supernate of the coculture system was determined by ELISA. Results MSCs overexpressed positive surface molecules CD29 and CD90, and low expression of hematopoietic cell surface marker CD45. BMSCs could significantly inhibit the proliferation and activation of HSCs, and promote apoptosis of HSCs. The concentration of HGF in the supernate was significantly higher in M-H group than that in the other groups. Conclusion BMSCs can promote the apoptosis of HSCs, inhibit the proliferation and activation of HSCs through paracrine HGF in the co-culture of BMSCs and HSCs.
引文
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[2] Milosavljevic N,Gazdic M,Simovic MB,et al. Mesenchymal stemcells attenuate liver fibrosis by suppressing Th17 cells[J]. Transpl Int,2018,31(1):102-115. doi:10.1111/tri.13023.
[3] Raafat N,Abdel Aal SM,Abdo FK,et al. Mesenchymal stem cells:In vivo therapeutic application ameliorates carbon tetrachlorideinduced liver fibrosis in rats[J]. Int J Biochem Cell Biol,2015,68:109-118. doi:10.1016/j.biocel.2015.09.003.
[4] Suk KT,Yoon J,Kim MY,et al. Transplantation with autologousbone marrow-derived mesenchymal stem cells for alcoholiccirrhosis:Phase 2 trial[J]. Hepatology,2016,64(6):2185-2197.doi:10.1002/hep.28693.
[5] Rengasamy M,Singh G,Fakharuzi NA,et al. Transplantation ofhuman bone marrow mesenchymal stromal cells reduces liverfibrosis more effectively than Wharton’s jelly mesenchymal stromalcells[J]. Stem Cell Res Ther,2017,8(1):143. doi:10.1186/s13287-017-0595-1.
[6]林楠,谢树杰,潘卫东,等.大鼠骨髓间质干细胞体内诱导肝星状细胞的凋亡[J].中国组织工程研究与临床康复,2010,14(10):1769-1774. Lin N,Xie SJ,Pan WD,et al. Rat bone marrowmesenchymal stem cells induce hepatic stellate cells apoptosis invivo[J]. Journal of Clinical Rehabilitative Tissue EngineeringResearch,2010,14(10):1769-1774.
[7] Forte G,Minieri M,Cossa P,et al. Hepatocyte growth factor effectson mesenchymal stem cells:proliferation, migration, anddifferentiation[J]. Stem Cells,2006,24(1):23-33. doi:10.1634/stemcells.2004-0176
[8] Lu T,Yang C,Sun H,et al. FGF4 and HGF promote differentiationof mouse bone marrow mesenchymal stem cells into hepatocytes viathe MAPK pathway[J]. Genet Mol Res,2014,13(1):415. doi:10.4238/2014.January.21.9.
[9] Bottaro DP,Rubin JS,Faletto DL,et al. Identification of thehepatocyte growth factor receptor as the c-met proto-oncogeneproduct[J]. Science,1991,251(4995):802-804. doi:10.1126/science.1846706.
[10]Horiguchi K,Hirano T,Ueki T,et al. Treating liver cirrhosis indogs with hepatocyte growth factor gene therapy via the hepaticartery[J]. J Hepatobiliary Pancreat Surg,2009,16(2):171-177.doi:10.1007/s00534-0 08-0029-7.
[11]Ohnishi S,Sumiyoshi H,Kitamura S,et al. Mesenchymal stem cellsattenuate cardiac fibroblast proliferation and collagen synthesisthrough paracrine actions[J]. FEBS Lett,2007,581(21):3961-3966. doi:10.1016/j.febslet.2007.07.028.
[12]Parekkadan B,van Poll D,Megeed Z,et al. Immunomodulation ofactivated hepatic stellate cells by mesenchymal stem cells[J].Biochem Biophys Res Commun,2007,363(2):247-252. doi:10.1016/j.bbrc.2007.05.150.
[13]韦柳萍,覃山羽,姜海行,等.大鼠骨髓间充质干细胞促进肝星状细胞凋亡的机制研究[J].中华肝脏病杂志,2014,22(3):223-227. Wei LP,Qin SY,Jiang HX,et al. Mechanism of bone marrow-derived mesenchymal stem cell-promoted apoptosis of hepaticstellate cells[J]. Chin J Hepatol,2014,22(3):223-227. doi:10.3760/cma.j.issn.1007-3418.2014.03.016.
[14]Wang PP,Xie DY,Liang XJ,et al. HGF and direct mesenchymalstem cells contact synergize to inhibit hepatic stellate cellsactivation through TLR4/NF-kB pathway[J]. PLoS One,2012,7(8):e43408. doi:10.1371/journal. pone.0043408.
[15]Ozaki I,Zhao G,MizutaI T,et al. Hepatocyte growth factor inducescollagenase(matrix metalloproteinase-1)via the transcriptionfactor Ets-1 in human hepatic stellate cell line[J]. J Hepatol,2002,36(2):169-178. doi:10.1016/S0168-8278(01)00245-8.
[16]方雪晴,张骏飞,宋海燕,等.脐带间充质干细胞移植对失代偿期乙型肝炎肝硬化患者免疫功能和转归的影响[J].中华肝脏病杂志,2016,24(12):907-910. Fang XQ,Zhang JF,Song HY,et al.Effect of umbilical cord mesenchymal stem cell transplantation onimmune function and prognosis of patients with decompensatedhepatitis B cirrhosis[J]. Chin J Hepatol,2016,24(12):907-910.doi:103760/cma.j.issn.1007-3418.2016.12.006.
[17]Wang L,Han Q,Chen H,et al. Allogeneic bone marrowmesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis[J]. Stem Cells Dev,2014,23(20):2482-2489. doi:10.1089/scd.2013.0500.
[18]Phinney DG, Pittenger MF. Concise review:MSC-Derivedexosomes for cell-free therapy[J]. Stem Cells,2017,35(4):851.doi:10.1002/stem.2575.
[19]Altanerova U,Jakubechova J,Repiska V,et al. Exosomes of humanmesenchymal stem/stromal/medicinal signaling cells.A review[J].Neoplasma,2017,64(6):809-815. doi:10.4149/neo_2017_601.
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[21]Li T,Yan Y,Wang B,et al. Exosomes derived from humanumbilical cord mesenchymal stem cells alleviate liver fibrosis[J].Stem Cells Dev,2013,22(6):845-854.