Scleraxis慢病毒转染人羊膜间充质干细胞促进兔腱-骨愈合  

Scleraxis lentivirus-transfected human amniotic mesenchymal stem cells promote tendon-bone healing in rabbits

作　　者：邹刚 张骏 尤奇 汤井沣 金瑛 杨继滨 朱喜忠 刘毅 Zou Gang;Zhang Jun;You Qi;Tang Jingfeng;Jin Ying;Yang Jibin;Zhu Xizhong;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2080-2086,共7页Chinese Journal of Tissue Engineering Research

基　　金：贵州省科技厅联合基金(黔科合LH字[2017]7105号)，项目负责人：邹刚~~

摘　　要：背景:人羊膜间充质干细胞来源广泛、免疫排斥低,具有多系分化潜能,研究证实Scleraxis基因能够诱导人羊膜间充质干细胞向韧带分化,加速腱-骨愈合进程。目的:探讨Scleraxis诱导人羊膜间充质干细胞分化后是否能够在体内促进兔腱-骨愈合,为临床腱-骨愈合提供新的思路和方向。方法:①经遵义医科大学附属医院伦理委员会批准,术前签署知情同意书,取健康足月产妇胎盘,经过2次胰酶消化分离培养人羊膜间充质干细胞,然后倒置显微镜下观察细胞形态,传代继续培养至第3代用于后续实验;②体外将携带有Scleraxis基因的慢病毒转染至人羊膜间充质干细胞,通过实时荧光定量PCR检测韧带相关基因的表达水平,免疫荧光检测韧带相关蛋白的表达水平;③将转染Scleraxis基因的人羊膜间充质干细胞注射到兔关节外腱-骨模型中,3个月后取标本观察腱-骨愈合情况。结果与结论:①传代至第3代的人羊膜间充质干细胞在倒置相差显微镜下呈长梭形、涡旋状贴壁生长;②第3代人羊膜间充质干细胞经过Scleraxis基因慢病毒感染24 h后表达绿色荧光,96 h荧光表达量最强且稳定;③CCK-8检测转染病毒后对细胞的生长速度无影响;④实时荧光定量PCR显示:慢病毒转染Scleraxis基因后,其mRNA表达量成倍的增加,并且韧带相关基因Ⅰ型胶原、Ⅲ型胶原、Fibronectin、Tenascin-C的mRNA表达量也明显提高;⑤免疫荧光结果显示:慢病毒转染Scleraxis基因后其韧带相关蛋白Ⅰ型胶原、Ⅲ型胶原、Fibronectin、Tenascin-C的表达水平提高;⑥动物体内实验证实:慢病毒转染Scleraxis基因后能够加速兔关节外腱-骨模型的腱-骨愈合。BACKGROUND: Human amniotic mesenchymal stem cells have a wide variety of sources, low immunogenicity, and multilineage differentiation potential. Studies have confirmed that Scleraxis gene can induce human amniotic mesenchymal stem cells to differentiate into ligaments and accelerate tendon-bone healing. OBJECTIVE: To explore whether Scleraxis induces human amniotic mesenchymal stem cells to promote tendon-bone healing in vivo in rabbits, providing new options for clinical treatment of tendon-bone healing. METHODS: The study protocol was approved by the Ethic Committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The healthy full-term maternal placenta was taken and cultured, and human amniotic mesenchymal stem cells were isolated and cultured by trypsin digestion twice. Then the morphology of the cells was observed under an inverted microscope, and the cells were further cultured until the third generation for subsequent experiments. The lentivirus carrying the Scleraxis gene was transfected into human amniotic mesenchymal stem cells in vitro. Expression levels of ligament-related genes were detected by real-time fluorescent quantitative PCR, and the expression levels of related proteins were detected by immunofluorescence. Human amniotic mesenchymal stem cells transfected with Scleraxis gene were injected into the extraarticular tendon-bone model of rats. After 3 months, specimens were taken to observe the tendon-bone healing. RESULTS AND CONCLUSION:(1) Human amniotic mesenchymal stem cells from passage to third generation showed long fusiform and vortex-like adherent growth under the inverted phase contrast microscope.(2) The third-generation human amniotic mesenchymal stem cells expressed green fluorescence after 24 hours of infection with the Scleraxis gene lentivirus, and the fluorescence expression was strong and stable.(3) Cell counting kit-8 findings indicated that lentivirus transfection of Scleraxis gene showed no influence on the

关 键 词：人羊膜间充质干细胞 SCLERAXIS 基因转染 慢病毒 腱-骨愈合 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]