基因修饰人BMSCs构建组织工程骨的实验研究  

EXPERIMENTAL STUDY ON TISSUE ENGINEERED BONES CONSTRUCTED BY HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE-MODIFIED HUMAN BONE MARROW MESENCHYMAL STEM CELLS

作　　者：俞莉敏[1] 马俊轩 于滨生[1] 

机构地区：[1]北京大学深圳医院脊柱外科.深圳市脊柱外科重点实验室,广东深圳518036

出　　处：《中国修复重建外科杂志》2016年第12期1512-1517,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：广东省医学科研基金资助项目(A2014654)~~

摘　　要：目的探索构建可稳定表达修饰基因人BMP-2(human BMP-2,hBMP-2)的细胞组织工程骨促进骨再生的可行性。方法从成人肌肉组织中以RT-PCR方法克隆hBMP-2基因全长,连接构建真核质粒载体,经脂质体转染人BMSCs(humanBMSCs,hBMSCs)。设定hBMP-2基因转染细胞组、空质粒载体转染细胞组以及同代细胞正常培养组,经G418筛选培养后,分别行细胞ALP比活性测定、细胞hBMP-2免疫组织化学染色、hBMP-2 mRNA表达水平的RT-PCR检测及细胞上清的hBMP-2分泌水平的免疫酶斑点(dot-ELISA)检测。然后将基因转染细胞与羟基磷灰石(hydroxyapatite,HA)材料进行复合培养,观察组织工程骨的构建情况。动物实验分4组,每组3只裸鼠,于裸鼠双侧背脊肌内分别植入hBMP-2基因转染细胞与HA材料复合培养体(A组),空载质粒转染细胞复合HA材料(B组)、单纯基因转染细胞悬液(C组)以及hBMP-2基因质粒加脂质体(D组)作为对照。4组裸鼠均于4周后取材、脱钙、切片,行HE染色及阿尔新蓝染色观察新骨形成情况。结果 hBMP-2基因转染细胞在培养48 h和3周时均可表达分泌外源基因hBMP-2,免疫组织化学染色呈阳性,且成骨分化的ALP比活性明显高于两对照组(P<0.05)。转染细胞能良好贴附于HA表面生长。动物实验中,A组3只裸鼠6侧背脊肌内4侧有较多新骨生成;B组3只裸鼠中的1只双侧背脊肌内有新骨生成;C组未发现新骨生成;D组1只裸鼠1侧背脊肌内发现少量成骨。结论 hBMP-2基因转染修饰的h BMSCs与HA复合培养构建的组织工程骨,可瞬时和稳定表达分泌hBMP-2,并能促进裸鼠肌肉组织内异位成骨。Objective To investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells （hBMSCs） expressing the transduced human bone morphogenetic protein 2 （hBMP-2） gene stably. Methods The full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome, hBMSCs were transfected by hBMP-2 gene （experimental group） and by empty plasmid （negative control group）, untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection, hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite （HA） and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone （group A, n=3）, empty plasmid transfected hBMSCs seeded on HA （group B, n=3）, hBMSCs suspension transfected by hBMP-2 gene （group C, n=3）, and hBMP-2 plasmids and lipidosome （group D, n=3） were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. Results At 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups （P〈0.05）. The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated

关 键 词：组织工程骨 BMSCS BMP-2 基因治疗 转染 

分 类 号：R68[医药卫生—骨科学]