骨形态发生蛋白2基因转染人羊水干细胞复合β-磷酸三钙支架促进骨再生  

Bone morphogenetic protein-2 gene-transfected human amniotic fluid-derived stem cells combinedwith β-tricalcium phosphate stents promote bone regeneration

作　　者：马超[1] 刘光旺[2] 张艳玲[1] 高娟[1] 郭含军[2] 曲新华[3] 翟赞京 谢幼专[3] 戴尅戎[3] 

机构地区：[1]东南大学医学院附属徐州市中心医院骨科,江苏徐州221009 [2]徐州医学院徐州临床学院 [3]上海交通大学医学院附属第九人民医院骨科

出　　处：《中华生物医学工程杂志》2014年第2期94-99,共6页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金面上项目（81071453）;江苏省中医药局项目（LZ13132）;江苏省科技厅临床诊疗技术攻关项目（BL2014026）;徐州市首届医学青年后备人才计划（2014006）;徐州市中心医院博士创新团队科技项目（XZB201236）

摘　　要：目的研究重组腺病毒介导的骨形态发生蛋白2（BMP-2）基因转染人羊水干细胞（hAFSC）的体外成骨分化，评价基因修饰的hAFSC负载于β-磷酸三钙（β-TCP）多孔支架后体内成骨能力。方法收集人工破膜后的中段羊水原代培养hAFSC，免疫磁珠分选CDl17／c-kit阳性的hAFSC，分别转染腺病毒介导的BMP-2或增强型绿色荧光蛋白（EGFP）基因，转染组转染BMP-2和EGFP基因、对照组转染无BMP-2编码序列的EGFP基因，空白组不做病毒转染。48h后荧光显微镜下观察细胞荧光表达及生长状况，应用流式细胞仪检测转染率。转染第7、14、21、28天采用ELISA检测各组hAFSC培养液中的BMP．2分泌量以确认转染后目的蛋白表达效果。各组hAFSC成骨诱导14d后进行碱性磷酸酶（ALP）染色，28d后进行茜素红染色评价成骨能力；成骨诱导3、7、14d后定量检测各组细胞ALP活性。20只8周龄裸小鼠随机分为Adv-hBMP-2-hAFSC-β-TCP组、Adv-EGFP-hAFSC-β-TCP组、hAFSC-β-TCP组和β-TCP组，每组5只，将细胞载体复合物植入各组裸小鼠股骨，8周后观察异位成骨组织学情况。结果hAFSC生长良好，Adv-hBMP-2或Adv-EGFP基因转染48h后荧光显微镜下见细胞贴壁良好，发出强绿色荧光，无死细胞产生，转染阳性率达（89．00±4．25）％。转染Adv-hBMP-2组hAFSC培养液上清中BMP-2第7、14、21、28天分别为（3．405±0．229）μg／L、（4．575±0．179）μg／L、（3．910±0．175）μg／L、（2．694±0．205）μg/L，第14天蛋白BMP-2分泌已达到高峰且第28天仍有蛋白表达，各时间点均明显高于对照组与空白组（均P〈0．05）。hAFSC成骨诱导14d后细胞相连成片，与对照组及空白组比较，ALP染色转染组染色阳性面积更大、阳性细胞数量更多。成骨诱导28d后茜素红染色见转染组细胞多中心聚集，伴大量红色钙结节形成，结节数量与大小明显优于对照组与空白组。成骨诱�Objective To study the in vitro osteoblastic differentiation of human amniotic fluid- derived stern cells （hAFSCs） transfected with bone morphogenetic protein-2 （BMP-2） gene, and to evaluate the in vivo bone formation ability of genetically modified hAFSCs when loaded onto porous β-tricalcium phosphate （β-TCP） stents. Methods hAFSCs were primarily cultured from mid- stream amniotic fluid collected after artificial rupture of amniotic sac. CD117/c- kit positive hAFSCs were obtained by using immunoselection with magnetic beads, and were then subjected to adenovirus-mediated transfection of BMP- 2 and enhanced green fluorescent protein （EGFP） （tranfection group）, or EGFP alone （control group） or no transfection （non- transfection group）. Forty- eight hours later, the hAFSCs were examined for EGFP expression and cell growth under fluorescent microscopy, and for rate of transfection by flow cytometry. At 7, 14, 21 and 28 days after transfection, ELISA was used to measure the level of BMP-2 in the cell culture of each group, so as to confirm the expression of target proteins. Osteogenesis ability was assessed with alkaline phosphatase （ALP） staining on day 14 and alizarin red staining on day 28. The activity of ALP was quantitatively assessed in all groups after 3, 7, and 14 days of osteogenic induction. A total of 20 8-week-old nude mice were randomized into four groups （n=5 each） to receive femoral implantation of different complex of hAFSCs and β-TCP stents ： Adv-hBMP-2-hAFSC-β-TCP, Adv-EGFP-hAFSC-β-TCP, hAFSC-β-TCP or β- TCP. Ectopic bone formation was evaluated and compared among these groups at week 8. Results The hAFSCs grew well. At 48 h after Adv-hBMP-2 or Adv-EGFP transfection, fluorescent microscopy showed that the hAFSCs appeared normally wall-adhering, shining with strong green fluorescence, with a positive rate of transfection being （89.00±4.25）%, and no cell death. The level of BMP-2 in the supernatant of hAFSCs culture was （3.405±0.229）μg/L on day

关 键 词：骨形态发生蛋白质类 羊水干细胞 基因 转染 β-磷酸三钙支架 骨再生 

分 类 号：R68[医药卫生—骨科学]