Yu Limin, Ma Junxuan, Yu Binsheng
Shenzhen Key Laboratory of Spine Surgery, Department of Spine Surgery, Peking University Shenzhen Hospital, Shenzhen Guangdong, 518036, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Dec 8;30(12):1512-1517. doi: 10.7507/1002-1892.20160313.
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.
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, =3), empty plasmid transfected hBMSCs seeded on HA (group B, =3), hBMSCs suspension transfected by hBMP-2 gene (group C, =3), and hBMP-2 plasmids and lipidosome (group D, =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.
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 (<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. 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.
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.
研究稳定表达转导人骨形态发生蛋白2（hBMP-2）基因的人骨髓间充质干细胞（hBMSCs）构建的细胞-组织工程骨的骨再生潜力。
通过RT-PCR从人肌肉组织中克隆全长hBMP-2基因，并连接到载体中构建真核表达系统。然后用脂质体将该基因表达系统转导至hBMSCs。hBMSCs分别用hBMP-2基因转染（实验组）和空质粒转染（阴性对照组），未转染hBMP-2的作为空白对照组。采用RT-PCR、斑点ELISA、免疫组织化学分析及碱性磷酸酶（ALP）活性检测，比较和评价转染后hBMP-2的表达和分泌情况。将经hBMP-2基因转染的hBMSCs接种于羟基磷灰石（HA）上培养4天构建hBMP-2基因修饰的组织工程骨，然后用倒置相差显微镜和扫描电子显微镜观察组织工程骨。将hBMP-2基因修饰的组织工程骨（A组，n = 3）、接种于HA上的空质粒转染hBMSCs（B组，n = 3）、hBMP-2基因转染的hBMSCs悬液（C组，n = 3）以及hBMP-2质粒和脂质体（D组，n = 3）植入裸鼠双侧背部肌肉。4周后通过HE染色和阿尔辛蓝染色检测成骨活性。
转染后48小时和3周，RT-PCR和斑点ELISA结果表明，转染的hBMSCs能稳定表达和分泌活性外源hBMP-2。免疫组织化学染色呈阳性，转染的hBMSCs中ALP活性显著高于两个对照组（P<0.05）。在倒置相差显微镜和扫描电子显微镜下，转染的hBMSCs在HA三维表面上附着生长良好。研究表明，A组6只裸鼠背部肌肉6个面中的4个面有明显的大量新骨形成。B组3只裸鼠中有1只双侧背部肌肉有一些新骨形成。C组未发现新骨形成。D组有1只裸鼠背部肌肉一侧有少量新骨形成。
hBMP-2基因修饰的hBMSCs与HA构建的组织工程骨能稳定表达和分泌活性hBMP2，并能有效促进裸鼠肌肉内新骨形成。
