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Title: [EXPERIMENTAL STUDY ON TISSUE ENGINEERED BONES CONSTRUCTED BY HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE-MODIFIED HUMAN BONE MARROW MESENCHYMAL STEM CELLS].
Author: Yu L, Ma J, Yu B.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2016 Dec 08; 30(12):1512-1517. PubMed ID: 29786344.
Abstract:
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 that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. CONCLUSIONS: The tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice. 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（human BMSCs，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hBMSCs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。.

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