人脱细胞羊膜支架促进Scleraxis修饰人羊膜间充质干细胞体外成韧带分化  

Human acellular amniotic membrane scaffold promotes ligament differentiation of human amniotic mesenchymal stem cells modified by Scleraxis in vitro

作　　者：邹刚 徐志 刘子铭 李豫皖 杨继滨 金瑛 张骏 葛振 刘毅 Zou Gang;Xu Zhi;Liu Ziming;Li Yuwan;Yang Jibin;Jin Ying;Zhang Jun;Ge Zhen;Liu Yi(Department of Orthopedics,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China;Zunyi Medical University-Rochester University Orthopedic Research Center,Zunyi 563000,Guizhou Province,China;Second Department of Orthopedics,Hospital of the Jingxian County,Jingxian County 242500,Anhui Province,China;Institute of Sports Medicine,Peking University Third Hospital,Beijing 100191,China)

机构地区：[1]遵义医科大学附属医院骨科,贵州省遵义市563000 [2]遵义医科大学-罗切斯特大学骨科研究中心,贵州省遵义市563000 [3]泾县医院骨二科,安徽省泾县242500 [4]北京大学第三医院运动医学研究所,北京市100191

出　　处：《中国组织工程研究》2021年第7期1037-1044,共8页Chinese Journal of Tissue Engineering Research

基　　金：贵州省科学技术基金项目(黔科合LH字[2017]7105号),项目负责人:邹刚。

摘　　要：背景:脱细胞羊膜为一种天然的生物材料支架,已广泛应用于组织工程的相关领域。Scleraxis基因在结缔组织如韧带等形成中具有重要的调控作用。目的:验证Scleraxis修饰人羊膜间充质干细胞与人脱细胞羊膜支架复合物在大鼠体内的生物相容性。方法:取足月产胎盘羊膜组织,采用化学-酶消法对人新鲜羊膜进行脱细胞处理。两步酶消化法从人新鲜羊膜分离人羊膜间充质干细胞。运用Scleraxis慢病毒转染第3代人羊膜间充质干细胞,然后与人脱细胞羊膜复合培养5,10,15 d时检测成韧带相关基因的mRNA表达。将18只SD大鼠随机分为3组:实验组将已经制备的细胞支架复合物植入大鼠背部皮下筋膜,阴性对照组在大鼠背部做一切口,不植入材料,空白对照组不做任何处理,术后1,4周取术区组织进行苏木精-伊红染色,术后4周取术区组织行CK蛋白免疫组化染色。结果与结论:①Scleraxis基因转染人羊膜间充质干细胞与人脱细胞羊膜复合培养可显著上调韧带分化相关基因Ⅰ型胶原、Ⅲ型胶原、纤维连接蛋白、细胞连接素C的mRNA表达水平;②术后1周时实验组大鼠局部组织可见新生肉芽组织,炎症反应较阴性对照组、空白对照组重;术后4周时实验组大鼠局部组织排列趋于整齐,肉芽组织减少,炎症明显消退;③术后4周时实验组CK蛋白表达阳性,局部组织排列整齐,羊膜组织周围已经有大量细胞附着;④结果表明,Scleraxis基因转染人羊膜间充质干细胞与人脱细胞羊膜支架共培养可促进人羊膜间充质干细胞体外成韧带分化,该细胞支架复合物在动物体内表现出较好的生物相容性。BACKGROUND:Acellular amniotic membrane is a natural biomaterial scaffold,which has been widely used in related fields of tissue engineering.Scleraxis gene plays an important regulatory role in the formation of connective tissues such as ligaments.OBJECTIVE:To verify the biocompatibility of Scleraxis modified human amniotic mesenchymal stem cells and human acellular amniotic membrane scaffold complex in rats.METHODS:The amniotic membrane tissues of the placenta at term were taken,and the fresh human amniotic membrane was decellularized by chemicalenzymatic digestion method.Two-step enzyme digestion method was used to separate human amniotic mesenchymal stem cells from human fresh amniotic membrane.The third-generation human amniotic mesenchymal stem cells were transfected with Scleraxis lentivirus,which were then cultured with human decellularized amniotic membrane for 5,10,and 15 days so as to detect mRNA expression of related genes.The 18 Sprague-Dawley rats were randomly divided into three groups.In the experimental group,the prepared cell scaffold complex was implanted into the subcutaneous fascia of the back of the rat.In the negative control group,an incision was made on the back of the rat,without implanting materials.In the blank control group,no treatment was performed.Hematoxylin-eosin staining was performed on the operation area tissues 1 and 4 weeks after surgery.Immunohistochemical staining of CK protein was performed on the operation area tissues 4 weeks after surgery.RESULTS AND CONCLUSION:(1)Scleraxis gene transfection of human amniotic mesenchymal stem cells and human acellular amniotic membrane compound culture could significantly up-regulate the mRNA expression levels of ligament differentiation-related genes type I collagen,type III collagen,fibronectin and cytocontin C.(2)The neonatal granulation tissue was seen in the local tissue of the experimental group at 1 week after surgery,and the inflammatory response was heavier than that of the negative control group and the blank control group.At 4

关 键 词：干细胞 间充质干细胞 羊膜 脱细胞 韧带 支架 基因 大鼠 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]