纳米级多孔负载人骨形成蛋白2基因修饰支架对前成骨细胞株分化的影响  

Effects of nano porous beta-tricalcium phosphate/collagen scaffold modified with human bone morphogenetic protein 2 gene on differentiation of MC3T3-E1 cell lines

作　　者：阮蔷 赵刚[1] 郭睿[1] 肖月[1] 李超 

机构地区：[1]佳木斯大学口腔医学院,黑龙江省佳木斯市154007

出　　处：《中国组织工程研究》2016年第38期5657-5663,共7页Chinese Journal of Tissue Engineering Research

基　　金：黑龙江省自然科学基金项目(H201487);佳木斯大学研究生创新项目(LZR2015_015)~~

摘　　要：背景:对于骨缺损,通常需要通过骨组织或成骨材料的移植或充填进行修复。切取自体骨组织会给患者带来额外的损伤和继发畸形,为此,新一代的成骨材料研究迫在眉睫。目的:构建含人骨形成蛋白2质粒DNA的β-磷酸三钙/胶原支架材料,并观察其对小鼠前成骨细胞MC3T3-E1分化的影响。方法:制备纳米级多孔β-磷酸三钙/胶原支架并负载含人骨形成蛋白2目的基因的质粒DNA形成基因修饰的支架材料。建立小鼠前成骨细胞细胞株MC3T3-E1与复合支架的体外培养体系。将其分为支架组和平皿组,再根据质粒的浓度每组再分为2个小组。复合培养后取样通过扫描电镜和光镜进行细胞形态学观察;诱导1,3,7,14 d行茜素红染色观察钙结节情况;实时荧光定量PCR检测细胞周期;诱导1,3,7,14 d观察成骨细胞相关基因Ⅰ型胶原αⅠ链基因、锌指结构转录因子、骨唾液酸蛋白的表达。结果与结论:(1)含人骨形成蛋白2为目的基因的质粒DNA修饰纳米β-磷酸三钙/Ⅰ型胶原溶液复合材料上细胞黏附数、分化数量及材料表面分布都高于单纯含人骨形成蛋白2为目的基因的质粒DNA(P<0.05);(2)通过用茜素红染色、实时荧光定量PCR检测,结果显示支架组对小鼠前成骨细胞MC3T3-E1成骨能力的影响优于平皿组;(3)结果提示,负载人骨形成蛋白2基因修饰的β-磷酸三钙/胶原支架材料将具有骨传导性的支架与具有生物活性的成骨因子复合,形成复合支架同时具备骨传导性和骨诱导性,是一种理想的骨缺损修复材料。BACKGROUND： Bone tissue transplantation or osteogenic material filling is after used for bone defect repair. To remove autologous bone tissues can lead to additional damage and secondary deformity, therefore, it is extremely urgent to search for a new osteogenic material. OBJECTIVE： To construct the porous β-tricalcium phosphate（β-TCP）/collagen scaffold modified with human bone morphogenetic protein 2（h BMP2） gene, and to observe its effects on differentiation of MC3T3-E1 cell lines. METHODS： The porous β-TCP/collagen scaffold modified with hB MP2 gene was prepared. Then in vitro culture system of MC3T3-E1 cell lines with composite scaffold was established. There were scaffold and plate groups, and each group was divided into two subgroups according to the different concentrations of plasmid. Samples were collected and observed morphologically by scanning electron microscope and light microscope after complex culture. After 1, 3, 7 and 14 days of induction, calcium nodules were observed through alizarin red staining, the cell cycle was detected by real-time PCR, and expressions of α I-chain collagen type I gene, Osterix and bone sialoprotein were observed. RESULTS AND CONCLUSION： The number of cells adhered, differentated and distributed on the composite scaffold was significantly higher than that of the single scaffold（P 0.05）. Alizarin red staining and real-time PCR detection showed that the osteogenesis ability of MC3T3-E1 cell lines in the scaffold group was stronger than that in the plate group. To conclude, the porous β-TCP/collagen scaffold modified with h BMP2 gene is an appropriate candidate for bone defect repair.

关 键 词：骨形态发生蛋白质类 胶原 细胞分化 组织工程 生物材料 骨生物材料 人骨形成蛋白2 β-磷酸三钙/胶原 分化 骨缺损 黑龙江省自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]