脱细胞羊膜支架复合Scleraxis慢病毒转染的人羊膜间充质干细胞促进兔腱-骨愈合  

Acellular amniotic membrane scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis lentivirus can promote tendon-bone healing in rabbits

作　　者：张骏 杨继滨 金瑛 邹刚 汤井沣 葛振 杨启帆 刘毅 Zhang Jun;Yang Jibin;Jin Ying;Zou Gang;Tang Jingfeng;Ge Zhen;Yang Qifan;Liu Yi(First Department of Orthopedics,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China)

机构地区：[1]遵义医科大学附属医院骨一科

出　　处：《中国组织工程研究》2020年第13期2061-2067,共7页Chinese Journal of Tissue Engineering Research

基　　金：贵州省科学技术基金项目(黔科合LH[2017]7015号)，项目负责人：邹刚~~

摘　　要：背景:脱细胞羊膜支架是一种天然支架,具有良好的生物相容性,已经广泛应用于组织工程的相关领域。Scleraxis能够促进人羊膜间充质干细胞向人韧带细胞分化,进而促进腱-骨愈合。目的:探讨脱细胞羊膜支架复合Scleraxis慢病毒转染的人羊膜间充质干细胞能否促进兔腱-骨愈合。方法:①体外分离培养人羊膜间充质干细胞,经过传代培养后观察细胞的形态;②体外构建Scleraxis慢病毒然后以最适感染复数转染第3代人羊膜间充质干细胞,q-PCR检测其转染效率;③用酶消化法制备脱细胞羊膜支架,然后体外将转染Scleraxis慢病毒的人羊膜间充质干细胞接种到脱细胞羊膜支架上面,鬼笔环肽染色观察细胞在支架上的生长情况;④将脱细胞羊膜支架复合转染Scleraxis慢病毒的人羊膜间充质干细胞包裹新西兰大白兔跟腱,然后移植到骨隧道内,观察其对腱-骨愈合的影响。结果与结论:①第3代人羊膜间充质干细胞呈贴壁生长,细胞生长状态良好;②Scleraxis慢病毒转染后96 h表达稳定的绿色荧光,Slclerxis的m RNA表达水平明显提高,说明转染成功;③脱细胞羊膜支架的上皮细胞基本消失,证明脱细胞比较彻底,同时其基底层完整保留,细胞外基质成分仍然存在;④通过鬼笔环肽染色发现细胞在脱细胞羊膜支架上生长良好,增殖未受到影响;⑤体内实验结果提示:人脱细胞羊膜支架复合Scleraxis慢病毒转染的人羊膜间充质干细胞具有促进腱-骨愈合的作用。BACKGROUND: Acellular amniotic membrane scaffold is a natural scaffold with good biocompatibility, which has been widely used in tissue engineering. Scleraxis can promote the differentiation of human amniotic mesenchymal stem cells into human ligament cells and promote tendon-bone healing. OBJECTIVE: To explore whether acellular amniotic membrane scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis can promote rabbit tendon-bone healing. METHODS:(1) Human amniotic mesenchymal stem cells were isolated and cultured in vitro. After passaged, the cell morphology was observed.(2) The Scleraxis lentivirus was constructed in vitro and then transfected into passage 3 human amniotic mesenchymal stem cells with optimal multiplicity of infection. The transfection efficiency was detected by q-PCR.(3) The acellular amniotic membrane scaffold was prepared by enzymatic digestion. Then the Scleraxis lentivirus-transfected cells were seeded on the acellular amniotic membrane scaffold in vitro. The cell growth on the scaffold was observed by phalloidin staining.(4) The New Zealand white rabbit tendon was covered with the acellular amniotic membrane scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis lentivirus, followed by implanted into the bone tunnel. The tendon-bone healing was detected. RESULTS AND CONCLUSION: The passage 3 human amniotic mesenchymal stem cells adhered well.(2) After transfected with Scleraxis lentivirus for 96 hours, stable green fluorescence was observed. The mRNA expression level of Sclerxis was significantly increased, indicating a success transfection. The epithelial cells of the acellular amniotic membrane scaffold disappeared, indicating a relatively complete decellularization. The basal layer remained intact, and the extracellular matrix component still existed. Phalloidin staining results revealed that the cells on the acellular amniotic membrane scaffold were in good adhesion and growth, and the cell proliferation was not affecte

关 键 词：羊膜间充质干细胞 脱细胞羊膜 脱细胞羊膜支架 腱-骨愈合 SCLERAXIS 组织工程 

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