体外共培养人脐带间充质干细胞对肝星状细胞增殖的影响
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摘要
目的探讨在体外共培养条件下,人脐带间充质干细胞对肝星状细胞增殖的调控机制。
方法采用Transwell间接共培养体系,实验组上层接种人脐带间充质干细胞(UCMSCs),下层接种肝星状细胞(HSCs-LX-2);对照组仅肝星状细胞单独培养。MTT法测肝星状细胞增殖抑制率；HOECHST染色法,直接观察肝星状细胞增殖抑制情况；流式分析技术检测各组肝星状细胞凋亡情况；ELISA法检测各实验组细胞上清液中TGF-β1蛋白的表达；荧光实时定量PCR检测各组肝星状细胞中TGF-β1、Smad3、Smad7mRNA的表达；Western blot法检测各组肝星状细胞TGF-β1、Smad3、Smad7蛋白的表达。
结果与对照组比较,实验组共培养24、48h,随着时间延长肝星状细胞增殖抑制率明显升高(P<0.05)；HOECHST染色可见实验共培养组肝星状细胞凋亡增多,且48h较24h更明显(P<0.05)；流式细胞分析示共培养组肝星状细胞凋亡多于对照组,且48h较24h更明显(P<0.05)；上清液中,共培养组TGF-β1较对照组少,随着时间增加,差异更明显(P<0.05)。共培养48h实验组TGF-β1及Smad3mRNA表达低于对照组,而Smad7mRNA表达高于对照组(P<0.05)。共培养24h、48hTGF-β1、Smad3蛋白的表达均低于对照组,Smad7蛋白表达高于对照组(P<0.05)。
结论人脐带间充质干细胞能够抑制肝星状细胞的增殖,促进其凋亡,其机制可能是通过下调TGF-β1、Smad3,上调Smad7蛋白表达而实现的。
Objective To explore the mechanism of human umbilical cord mesenchymal stem cells (UCMSCs) to regulate the proliferation of hepatic stellate cells (HSCs-LX2) under co-culture in vitro.
Methods Human UCMSCs and HSCs in the experimental group were cultured in the plastic culture plate(6holes) to establish the upper and lower double-cell co-culture system. HSCs were cultured alone as negative control group. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry, respectively. Cell supernatants were harvested to determine the concentration of transforming growth factor betal (TGF-β1) by enzyme-linked immunosorbent assay (ELISA). The TGF-β1, Smad3and Smad7mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of TGF-β1, Smad3and Smad7protein by Western blot..
Results HSCs co-cultured with UCMSCs significantly inhibited HSC proliferation compared with the negative control group at24,48hours(P<0.05); Flow cytometry showed that HSCs apoptosis of co-culture group was increased compared with negative control group, and48h is more obvious (P<0.05). The concentrations of TGF-β1in co-culture supernatants in the experimental group was significantly lower than those in the supernatants of HSCs cultured alone, and48h is more obvious (P<0.05). After HSCs co-cultured with BMSCs for48hours, the expression of TGF-β1, Smad3mRNA and protein was reduced and the expression of Smad7mRNA and protein was increased compared with negative control group (P<0.05).
Conclusion UCMSCs inhibited the proliferation of HSCs, possibly through inhibiting the TGF-β1and Smad3expression, increasing Smad7protein expression.
方法采用Transwell间接共培养体系,实验组上层接种人脐带间充质干细胞(UCMSCs),下层接种肝星状细胞(HSCs-LX-2);对照组仅肝星状细胞单独培养。MTT法测肝星状细胞增殖抑制率；HOECHST染色法,直接观察肝星状细胞增殖抑制情况；流式分析技术检测各组肝星状细胞凋亡情况；ELISA法检测各实验组细胞上清液中TGF-β1蛋白的表达；荧光实时定量PCR检测各组肝星状细胞中TGF-β1、Smad3、Smad7mRNA的表达；Western blot法检测各组肝星状细胞TGF-β1、Smad3、Smad7蛋白的表达。
结果与对照组比较,实验组共培养24、48h,随着时间延长肝星状细胞增殖抑制率明显升高(P<0.05)；HOECHST染色可见实验共培养组肝星状细胞凋亡增多,且48h较24h更明显(P<0.05)；流式细胞分析示共培养组肝星状细胞凋亡多于对照组,且48h较24h更明显(P<0.05)；上清液中,共培养组TGF-β1较对照组少,随着时间增加,差异更明显(P<0.05)。共培养48h实验组TGF-β1及Smad3mRNA表达低于对照组,而Smad7mRNA表达高于对照组(P<0.05)。共培养24h、48hTGF-β1、Smad3蛋白的表达均低于对照组,Smad7蛋白表达高于对照组(P<0.05)。
结论人脐带间充质干细胞能够抑制肝星状细胞的增殖,促进其凋亡,其机制可能是通过下调TGF-β1、Smad3,上调Smad7蛋白表达而实现的。
Objective To explore the mechanism of human umbilical cord mesenchymal stem cells (UCMSCs) to regulate the proliferation of hepatic stellate cells (HSCs-LX2) under co-culture in vitro.
Methods Human UCMSCs and HSCs in the experimental group were cultured in the plastic culture plate(6holes) to establish the upper and lower double-cell co-culture system. HSCs were cultured alone as negative control group. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry, respectively. Cell supernatants were harvested to determine the concentration of transforming growth factor betal (TGF-β1) by enzyme-linked immunosorbent assay (ELISA). The TGF-β1, Smad3and Smad7mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of TGF-β1, Smad3and Smad7protein by Western blot..
Results HSCs co-cultured with UCMSCs significantly inhibited HSC proliferation compared with the negative control group at24,48hours(P<0.05); Flow cytometry showed that HSCs apoptosis of co-culture group was increased compared with negative control group, and48h is more obvious (P<0.05). The concentrations of TGF-β1in co-culture supernatants in the experimental group was significantly lower than those in the supernatants of HSCs cultured alone, and48h is more obvious (P<0.05). After HSCs co-cultured with BMSCs for48hours, the expression of TGF-β1, Smad3mRNA and protein was reduced and the expression of Smad7mRNA and protein was increased compared with negative control group (P<0.05).
Conclusion UCMSCs inhibited the proliferation of HSCs, possibly through inhibiting the TGF-β1and Smad3expression, increasing Smad7protein expression.
引文
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[24]吴晓玲,曾维政,蒋明德,秦建平,徐辉.肝纤维化大鼠肝组织Smads基因表达状况及意义世界华人消化杂志2008；16：1037-1041
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[28]Liu C, Gaca MD, Swensen ES. Smads2 and 3 are diferentially activated by transforming growth factor-beta (TGF-beta)in quiescent and activated hepatic stellate cels. J Biol Chem 2003; 278:11721-11728
[29]Uemura M, Swenson ES, Gaca MD. Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization. Mol Biol Cell 2005; 16: 4214-4224
[30]Chen YW, Wu JX. Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor- beta signaling. World J Gastroenterol 2005; 21:2922-2926
[31]徐燕,李长虹,孟恒星,郝牧,邱录贵.中国组织工程研究与临床康复2009；13：6289-6294
[32]Cho KA, Lim GW, Joo SY. Transplantation of bone marrow cells reduces CC14 induced liver fibrosis in mice. Liver Int 2011; 31:932-939
[33]Kuo TK, Hung SP, Chuang CH. Stem cell therapy for liver disease:parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 2008; 134: 2111-2121
[34]Shi MX, Fan g BJ, Liao LM. Flkl+Mesenchyanal stem cells ameliorate carbon tetrachloride induced liver fibrosis in mice. Sheng Wu Gong Cheng Xue Bao 2005; 21:396-401
[35]Zhao DC, Le I JX, Chen R. Bone marrow derived mesenchymal stem cells protect against experimental liver fibrosis in rat. World J Gastroenterol 2005; 14:3431-3440
[36]Terai S, Ishikawa T, Omori K. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24:2292-2298
[37]姚鹏,胡大荣,王帅.自体骨髓干细胞移植治疗慢性肝衰竭研究.肝脏2005;3：171-173
[38]Nikeghbalian S, Pournasr B, Aghdami N. Autologous transplantation of bone marrow derived mononuclear and CD 133+ cells in patients with decompensated cirrhosis. Arch Iran Med 2011; 14:12-17
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[2]Minguell JJ, El'ices A, Conget P.Mesenehymal stem cell. ExpMed 2001; 226(6):507-520
[3]吕璐璐,宋永平,魏旭东,房伯俊,张艳莉,李玉富.人脐带和骨髓源间充质干细胞生物学特征的对比研究.中国实验血液学杂志2008；16：140-146
[4]Kocabayoglu P, Friedman SL. Cellular basis of hepatic fibrosis and its role in inflammation and cancer. Front Biosci (Schol Ed) 2013; 51:217-30
[5]Yoshida K, Matsuzaki K. Differential Regulation of TGF-β/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries. Front Physiol 2012; 3:53
[6]Luk JM, Wang PP. Lee CK, Wang JH, Fan ST. Hepatic potential of bone marrow stromal cells:development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods 2005; 305:39-47
[7]陈念,俞悦,李军.骨髓间充质干细胞移植在急性肝损伤大鼠肝脏内定制能力的实验研究.南京医科大学学报(自然科学版)2007；27：684-688
[8]胡义,王庆林.肝纤维化发病机制的研究进展.国际病理科学与临床杂志2006；27：48-52
[9]Breitkopf K, Haas S, Wiereinska E. Anti-TGF -beta strategies for the treatment of chronic liver disease. Alcohol Clin Exp Res 2005; 29:121S-131S
[10]Sirli R, Sporea I, Bota S, Ratiu I. Liver elastography for the diagnosis of portal hypertension in patients with liver cirrhosis. Med Ultrason 2012; 14:225-30
[11]Henderson NC, Forbes SJ. Hepatic fibrogenesis:from within and outwith. Toxicology 2008; 254:130-135.
[12]陈江,张荣华,吴蕙岭,夏勇,陈玉满,郑云燕.实验性肝纤维化形成和逆转过程中MMP-2及其抑制物TIMP-2表达的动态研究.中国卫生检验杂志2010；10：2401-2403
[13]金英姬,金仁顺,朴东明.四氯化碳诱发大鼠肝纤维化自发逆转中库普弗细胞与星状细胞的分布.世界华人消化杂志2007；15：2453-2457
[14]刘景章,张鹏,张金江,李兴杰,王首星.肝纤维化组织中RECK和MMP-2的表达及意义研究.临床合理用药杂志2013；3：2-4.
[15]李庆方,高爱荣.慢性乙肝组织中MMP-9与CD_(68)表达及临床意义.山东医药2008；38：78-79
[16]丁宁.肝纤维化形成机制的研究进展.临床肝胆病杂志2009；25：73-77
[17]杨悦杰,黄芬.肝星状细胞及相关细胞因子在肝纤维化行程中的作用.世界华人消化杂志2007；15：2885-2890
[18]周长升,王效民.肝星状细胞与肝癌的研究进展.肝胆外科杂志2009；17：395-397
[19]李婧,蒋炜.肝星状细胞的免疫学特性.复旦学报(医学版)2010；37：364-367
[20]Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-218
[21]李才,张一宁,牛俊奇.器官纤维化基础与临床.人民卫生出版社2003；5：49-51
[22]Nie QH, Duan GR, Luo XD, et al. Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis[J]. World J Gastroenterol,2004,10:86-90.
[23]Tahashi Y, M atsuzaki K. Date M Yoshida K Furukawa F Sugano Y Matsushita M Himeno Y Inagaki Y, Inoue K. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology 2002; 35:49-61
[24]吴晓玲,曾维政,蒋明德,秦建平,徐辉.肝纤维化大鼠肝组织Smads基因表达状况及意义世界华人消化杂志2008；16：1037-1041
[25]Breitkopf K, Haas S, Wiereinska E. Anti-TGF--beta strategies for the treatment of chronic liver disease. Alcohol Clin Exp Res 2005; 29:121S-131S
[26]Wang QL, Tao YY, Yuan JL, Shen L, Liu CH.Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro.BMC Cell Biol 2010; 11:31-32
[27]Yang L, Inokuchi S, Roh YS, Song J, Loomba R, Park EJ, Seki E.Transforming Growth Factor-β Signaling in Hepatocytes Promotes Hepatic Fibrosis and Carcinogenesis in Mice With Hepatocyte-Specific Deletion of TAK1. Gastroenterology 2013; 4:5082-5085
[28]Liu C, Gaca MD, Swensen ES. Smads2 and 3 are diferentially activated by transforming growth factor-beta (TGF-beta)in quiescent and activated hepatic stellate cels. J Biol Chem 2003; 278:11721-11728
[29]Uemura M, Swenson ES, Gaca MD. Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization. Mol Biol Cell 2005; 16: 4214-4224
[30]Chen YW, Wu JX. Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor- beta signaling. World J Gastroenterol 2005; 21:2922-2926
[31]徐燕,李长虹,孟恒星,郝牧,邱录贵.中国组织工程研究与临床康复2009；13：6289-6294
[32]Cho KA, Lim GW, Joo SY. Transplantation of bone marrow cells reduces CC14 induced liver fibrosis in mice. Liver Int 2011; 31:932-939
[33]Kuo TK, Hung SP, Chuang CH. Stem cell therapy for liver disease:parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 2008; 134: 2111-2121
[34]Shi MX, Fan g BJ, Liao LM. Flkl+Mesenchyanal stem cells ameliorate carbon tetrachloride induced liver fibrosis in mice. Sheng Wu Gong Cheng Xue Bao 2005; 21:396-401
[35]Zhao DC, Le I JX, Chen R. Bone marrow derived mesenchymal stem cells protect against experimental liver fibrosis in rat. World J Gastroenterol 2005; 14:3431-3440
[36]Terai S, Ishikawa T, Omori K. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24:2292-2298
[37]姚鹏,胡大荣,王帅.自体骨髓干细胞移植治疗慢性肝衰竭研究.肝脏2005;3：171-173
[38]Nikeghbalian S, Pournasr B, Aghdami N. Autologous transplantation of bone marrow derived mononuclear and CD 133+ cells in patients with decompensated cirrhosis. Arch Iran Med 2011; 14:12-17
[39]吴理茂,李连达,刘红.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤实验研究.中国工程科学2004;6：34—42
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