脂肪间充质干细胞复合多孔壳聚糖微球修复大鼠肝损伤
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摘要
背景:随着干细胞治疗及组织工程的发展,利用生物相容性好的生物高分子材料结合脂肪间充质干细胞构建肝脏组织工程微单元成为治疗肝脏疾病的新策略。目的:探讨脂肪间充质干细胞复合多孔壳聚糖微球对急性肝损伤的修复能力。方法:将SD乳鼠脂肪间充质干细胞与多孔壳聚糖微球在体外复合培养,分别在培养第1,3,5天观察细胞在微球上增殖情况。取36只SD大鼠(北京市维通利华实验技术有限公司提供),随机分为3组(n=12):空白对照组,腹腔注射生理盐水;急性肝损伤组,腹腔注射10%四氯化碳-橄榄油;脂肪间充质干细胞-多孔壳聚糖微球组,腹腔注射10%四氯化碳-橄榄油,于注射后第1天通过手术将脂肪间充质干细胞-多孔壳聚糖微球平铺于肝脏表面。分别于注射后第1,2,3,7天进行大体、血清学及组织学评价。结果与结论:(1)脂肪间充质干细胞与多孔壳聚糖微球复合后第1,3,5天,经过活/死染色观察到细胞可在微球上正常生长(死细胞数量远远少于活细胞数量),且细胞在不断增殖;(2)注射后第1,2,3,7天,脂肪间充质干细胞-多孔壳聚糖微球组的大体形态、肝功能及组织学评价均优于急性肝损伤组,从第3天开始脂肪间充质干细胞-多孔壳聚糖微球组与空白对照组已无明显差异;(3)结果表明,将脂肪间充质干细胞-多孔壳聚糖微球移植入肝脏表面可加速肝损伤的修复。
BACKGROUND:With the development of stem cell therapy and tissue engineering,adipose-derived mesenchymal stem cells combined with biocompatible scaffolds have been introduced to constructing liver tissue engineering micro-units as a new strategy for the treatment of liver diseases.OBJECTIVE:To investigate the repairing ability of adipose-derived mesenchymal stem cells combined with porous chitosan microspheres on acute liver injury.METHODS:Adipose-derived mesenchymal stem cells from neonatal Sprague-Dawley rats and porous chitosan microspheres were co-cultured in vitro to observe the proliferation of adipose-derived mesenchymal stem cells on the microspheres on the 1~(st),3~(rd) and 5~(th) days.Thirty-six Sprague-Dawley rats(provided by Beijing Vital River Laboratory Animal Technology Co.,Ltd.in China)were randomly divided into three groups(n=12 per group):control group was intraperitoneally injected with normal saline;acute liver injury group was intraperitoneally administered with 10% carbon tetrachloride-olive oil;and cell-microsphere group was intraperitoneally given 10% carbon tetrachloride-olive oil and then adipose-derived mesenchymal stem cells-porous chitosan microspheres were implanted into the liver surface on the 1~(st) day after injection.Gross,serological and histological evaluations were performed on the 1~(st),2~(nd),3~(rd) and 7~(th) days.RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells-porous chitosan microspheres were stained with live/dead staining on the 1~(st),3~(rd) and 5~(th) days.The cells grew normally on the microspheres as the number of dead cells was much less than that of living cells,and the cells proliferated continuously over time.The gross score,liver function score and histological score of the cell-microsphere group were higher than those of the acute liver injury group on the 1~(st),2~(nd),3~(rd) and 7~(th) days.There was no significant difference between the cell-microsphere group and control group since the 3~(rd) day.All these experimental findings indicate that adipose-derived mesenchymal stem cells-porous chitosan microspheres implanted onto the liver surface can accelerate the repair of liver injury.
BACKGROUND:With the development of stem cell therapy and tissue engineering,adipose-derived mesenchymal stem cells combined with biocompatible scaffolds have been introduced to constructing liver tissue engineering micro-units as a new strategy for the treatment of liver diseases.OBJECTIVE:To investigate the repairing ability of adipose-derived mesenchymal stem cells combined with porous chitosan microspheres on acute liver injury.METHODS:Adipose-derived mesenchymal stem cells from neonatal Sprague-Dawley rats and porous chitosan microspheres were co-cultured in vitro to observe the proliferation of adipose-derived mesenchymal stem cells on the microspheres on the 1~(st),3~(rd) and 5~(th) days.Thirty-six Sprague-Dawley rats(provided by Beijing Vital River Laboratory Animal Technology Co.,Ltd.in China)were randomly divided into three groups(n=12 per group):control group was intraperitoneally injected with normal saline;acute liver injury group was intraperitoneally administered with 10% carbon tetrachloride-olive oil;and cell-microsphere group was intraperitoneally given 10% carbon tetrachloride-olive oil and then adipose-derived mesenchymal stem cells-porous chitosan microspheres were implanted into the liver surface on the 1~(st) day after injection.Gross,serological and histological evaluations were performed on the 1~(st),2~(nd),3~(rd) and 7~(th) days.RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells-porous chitosan microspheres were stained with live/dead staining on the 1~(st),3~(rd) and 5~(th) days.The cells grew normally on the microspheres as the number of dead cells was much less than that of living cells,and the cells proliferated continuously over time.The gross score,liver function score and histological score of the cell-microsphere group were higher than those of the acute liver injury group on the 1~(st),2~(nd),3~(rd) and 7~(th) days.There was no significant difference between the cell-microsphere group and control group since the 3~(rd) day.All these experimental findings indicate that adipose-derived mesenchymal stem cells-porous chitosan microspheres implanted onto the liver surface can accelerate the repair of liver injury.
引文
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[24]Li Y,Hermanson DL,Moriarity BS,et al.Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.Cell Stem Cell.2018;23(2):181-192.e5.
[25]Fujita Y,Kawamoto A.Stem cell-based peripheral vascular regeneration.Adv Drug Deliv Rev.2017;120:25-40.
[26]Perico L,Morigi M,Rota C,et al.Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.Nat Commun.2017;8(1):983.
[27]Soliman MG,Mansour HA,Hassan WA,et al.Mesenchymal stem cells therapeutic potential alleviate lipopolysaccharide-induced acute lung injury in rat model.J Biochem Mol Toxicol.2018:e22217.doi:10.1002/jbt.22217.[Epub ahead of print]
[28]Jiang W,Tan Y,Cai M,et al.Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl4-Induced Liver Injury through Antioxidant Effect.Stem Cells Int.2018;2018:6079642.
[29]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.
[30]Padalhin AR,Lee BT.Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material.ASAIO J.2018 Aug25.doi:10.1097/MAT.0000000000000850.[Epub ahead of print]
[31]Shi D,Zhang J,Zhou Q,et al.Quantitative evaluation of human bone mesenchymal stem cells rescuing fulminant hepatic failure in pigs.Gut.2017;66(5):955-964.
[2]Kadota Y,Yagi H,Inomata K,et al.Mesenchymal stem cells support hepatocyte function in engineered liver grafts.Organogenesis.2014;10(2):268-277.
[3]An SY,Han J,Lim HJ,et al.Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells.Tissue Cell.2014;46(2):127-135.
[4]Ortiz LA,Dutreil M,Fattman C,et al.Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury.Proc Natl Acad Sci U S A.2007;104(26):11002-11007.
[5]Gilsanz C,Aller MA,Fuentes-Julian S,et al.Adipose-derived mesenchymal stem cells slow disease progression of acute-on-chronic liver failure.Biomed Pharmacother.2017;91:776-787.
[6]Nicolas CT,Wang Y,Nyberg SL.Cell therapy in chronic liver disease.Curr Opin Gastroenterol.2016;32(3):189-194.
[7]Xu L,Wang S,Sui X,et al.Mesenchymal Stem Cell-Seeded Regenerated Silk Fibroin Complex Matrices for Liver Regeneration in an Animal Model of Acute Liver Failure.ACS Appl Mater Interfaces.2017;9(17):14716-14723.
[8]Wiebe J,Nef HM,Hamm CW.Current status of bioresorbable scaffolds in the treatment of coronary artery disease.J Am Coll Cardiol.2014;64(23):2541-2551.
[9]Malcor JD,Juskaite V,Gavriilidou D,et al.Coupling of a specific photoreactive triple-helical peptide to crosslinked collagen films restores binding and activation of DDR2 and VWF.Biomaterials.2018;182:21-34.
[10]Subbarayan R,Girija DM,Rao SR.Human umbilical cord tissue stem cells and neuronal lineages in an injectable caffeic acid-bioconjugated gelatin hydrogel for transplantation.J Cell Physiol.2018 Aug 24.doi:10.1002/jcp.26834.[Epub ahead of print]
[11]Liu L,You Z,Yu H,et al.Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis.Nat Mater.2017;16(12):1252-1261.
[12]Etter JN,Karasinski M,Ware J,et al.Dual-crosslinked homogeneous alginate microspheres for mesenchymal stem cell encapsulation.J Mater Sci Mater Med.2018;29(9):143.
[13]Chander V,Singh AK,Gangenahalli G.Cell encapsulation potential of chitosan-alginate electrostatic complex in preventing natural killer and CD8+cell-mediated cytotoxicity:an in vitro experimental study.JMicroencapsul.2018:1-27.
[14]Wang Y,Yuan X,Yu K,et al.Fabrication of nanofibrous microcarriers mimicking extracellular matrix for functional microtissue formation and cartilage regeneration.Biomaterials.2018;171:118-132.
[15]Friedenstein AJ,Chailakhjan RK,Lalykina KS.The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells.Cell Tissue Kinet.1970;3(4):393-403.
[16]Fan CG,Tang FW,Zhang QJ,et al.Characterization and neural differentiation of fetal lung mesenchymal stem cells.Cell Transplant.2005;14(5):311-321.
[17]Caplan AI,Correa D.The MSC:an injury drugstore.Cell Stem Cell.2011;9(1):11-15.
[18]Manuguerra-GagnéR,Boulos PR,Ammar A,et al.Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment.Stem Cells.2013;31(6):1136-1148.
[19]Guo DB,Zhu XQ,Li QQ,et al.Efficacy and mechanisms underlying the effects of allogeneic umbilical cord mesenchymal stem cell transplantation on acute radiation injury in tree shrews.Cytotechnology.2018 Jul 31.doi:10.1007/s10616-018-0239-z.[Epub ahead of print]
[20]Chang SH,Huang HH,Kang PL,et al.In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering.Acta Biomater.2017;63:261-273.
[21]Hoyer DP,Munteanu M,Canbay A,et al.Liver transplantation for acute liver failure:are there thresholds not to be crossed.Transpl Int.2014;27(6):625-633.
[22]Pan XN,Zheng LQ,Lai XH.Bone marrow-derived mesenchymal stem cell therapy for decompensated liver cirrhosis:a meta-analysis.World JGastroenterol.2014;20(38):14051-14057.
[23]Kumari S,Vermeulen S,van der Veer B,et al.Shaping Cell Fate:Influence of Topographical Substratum Properties on Embryonic Stem Cells.Tissue Eng Part B Rev.2018;24(4):255-266.
[24]Li Y,Hermanson DL,Moriarity BS,et al.Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.Cell Stem Cell.2018;23(2):181-192.e5.
[25]Fujita Y,Kawamoto A.Stem cell-based peripheral vascular regeneration.Adv Drug Deliv Rev.2017;120:25-40.
[26]Perico L,Morigi M,Rota C,et al.Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.Nat Commun.2017;8(1):983.
[27]Soliman MG,Mansour HA,Hassan WA,et al.Mesenchymal stem cells therapeutic potential alleviate lipopolysaccharide-induced acute lung injury in rat model.J Biochem Mol Toxicol.2018:e22217.doi:10.1002/jbt.22217.[Epub ahead of print]
[28]Jiang W,Tan Y,Cai M,et al.Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl4-Induced Liver Injury through Antioxidant Effect.Stem Cells Int.2018;2018:6079642.
[29]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.
[30]Padalhin AR,Lee BT.Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material.ASAIO J.2018 Aug25.doi:10.1097/MAT.0000000000000850.[Epub ahead of print]
[31]Shi D,Zhang J,Zhou Q,et al.Quantitative evaluation of human bone mesenchymal stem cells rescuing fulminant hepatic failure in pigs.Gut.2017;66(5):955-964.