人羊膜间充质干细胞与人脱细胞羊膜支架的生物相容性  

Biocompatibility of human amniotic mesenchymal stem cells with human acellular amniotic membrane scaffold

作　　者：杨继滨 朱喜忠 熊华章[1] 李豫皖[1] 金瑛[1] 刘毅[1] Yang Ji-bin;Zhu Xi-zhong;Xiong Hua-zhang;Li Yu-wan;Jin Ying;Liu Yi(Department of Joint Surgery,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China)

机构地区：[1]遵义医学院附属医院关节外科,贵州省遵义市563000

出　　处：《中国组织工程研究》2018年第5期742-747,共6页Chinese Journal of Tissue Engineering Research

基　　金：贵州省科技厅联合基金(黔科合LH字[2016]7477号)~~

摘　　要：背景:前交叉韧带损伤越来越多见,传统治疗方式主要是前交叉韧带重建,随着组织工程研究在临床广泛运用,韧带组织工程治疗前交叉韧带损伤已成为目前研究的热点。目的:探讨人羊膜间充质干细胞和人脱细胞羊膜支架的体外生物相容性。方法:采用酶消化法获得人羊膜间充质干细胞并对其进行鉴定。采用酶消化法和化学去污法对新鲜人羊膜进行脱细胞处理,以苏木精-伊红染色以及免疫荧光染色检测细胞成分移除情况以及细胞外基质破坏情况。取第3代人羊膜间充质干细胞,分别使用人脱细胞羊膜浸提液(实验组)与普通L-DMEM/F12培养基(对照组)培养,CCK-8法检测两组细胞增殖能力。人羊膜间充质干细胞与人脱细胞羊膜共培养14 d后,扫描电镜与倒置显微镜观察人脱细胞羊膜上的细胞黏附及生长情况。结果与结论:①倒置相差显微镜观察显示人羊膜间充质干细胞呈漩涡状、贴壁生长,高表达波形蛋白,低表达CK-19,具有成骨、成脂、成软骨诱导分化能力;②苏木精-伊红染色显示人脱细胞羊膜上皮细胞及间充质干细胞已完全移除,细胞外基质成分无明显破坏;免疫荧光染色显示Ⅰ型胶原、Ⅲ型胶原均呈阳性表达;③人脱细胞羊膜浸提液对人羊膜间充质干细胞无细胞毒性,不影响其增殖活性;④倒置相差显微镜及扫描电镜均显示人脱细胞羊膜可以促进人羊膜间充质干细胞增殖及黏附,二者具有良好的生物相容性。BACKGROUND:Anterior cruciate ligament(ACL)injury is commonly seen,and mainly treated with ACL reconstruction.Ligament tissue engineering treatment for ACL injury has become the current focus due to the extensive use of tissue engineering techniques in clinical practice.OBJECTIVE:To investigate the biocompatibility of human amniotic mesenchymal stem cells(hAMSCs)and human acellular amniotic membrane(HAAM)scaffolds in vitro.METHODS:hAMSCs were isolated using enzyme digestion and then identified.The fresh human amniotic membrane was subjected to decellularization using enzymatic digestion and chemical decontamination.Hematoxylin-eosin staining and immunofluorescence were used to detect the removal of cell components and destruction of the extracellular matrix.hAMSCs were cultured with HAAM extract(experimental group)and normal L-DMEM/F12 medium(control group).Cell proliferation was detected by cell counting kit-8.Cell adhesion and growth were observed by scanning electron microscope and inverted microscope at 14 days after co-culture with HAAM.RESULTS AND CONCLUSION:hAMSCs exhibited a spiral,adherent growth under the inverted phase contrast microscope;and the cells highly expressed vimentin and lowly expressed CK-19,with osteogenic,adipogenic,and chondrogenic potential.Hematoxylin-eosin staining showed that human acellular amniotic epithelial cells and mesenchymal stem cells were completely removed and the extracellular matrix had no obvious damage.Immunofluorescence staining showed positive expression of type I collagen and type III collagen.The HAAM extract had no cytotoxicity to hAMSCs and exerted no effect on its proliferative activity.HAAM could promote the proliferation and adhesion of hAMSCs after incubation under the inverted microscope and scanning electron microscope.These findings indicate that HAAM has good biocompatibility and is favorable for the proliferation of hAMSCs.

关 键 词：人羊膜间充质干细胞 人脱细胞羊膜 支架 干细胞 组织工程 生物相容性 韧带损伤 前交叉韧带 

分 类 号：R394.2[医药卫生—医学遗传学]