目的 构建骨形成蛋白2(bone morphogenetic proteins 2,BMP2)绿色荧光融合蛋白pEGFP-N1-BMP2真核表达质粒,用其在体外转染犬牙髓细胞(dog dental pulp cells,DDPCs),利用构建的种子细胞BMP2-DDPCs与异种烧结骨(xenogeneic bone ceramice,XBC)复合,检测BMP2-DDPCs增殖活性,扫描电镜观察BMP2-DDPCs在异种烧结骨中的生长情况,评价异种烧结骨作为支架材料的可行性,为将人BMP2基因转染的牙髓成纤维细胞与异种烧结骨复合进行牙体修复的研究奠定基础.方法 构建pEGFP-N1-BMP2真核表达质粒,采用阳离子脂质体转染法将BMP2基因转染体外培养的犬牙髓细胞,将构建的种子细胞BMP2-DDPCs与异种烧结骨复合,MTT法检测BMP2-DDPCs增殖活性以及应用扫描电镜观察BMP2-DDPCs在异种烧结骨中的生长情况.结果 成功构建pEGFP-N1-BMP2真核表达质粒,并成功转染犬牙髓细胞.BMP2-DDPCs复合异种烧结骨组的细胞增殖活性与单层贴壁培养BMP2-DDPCs组的细胞增殖情况差异无统计学意义(P＞0.05).说明异种烧结骨上黏附的牙髓细胞增殖情况良好.扫描电镜观察异种烧结骨上BMP2-DDPCs黏附、生长的情况,可见支架材料的孔径为100～600μm,材料表面粗糙,利于细胞的黏附生长;孔隙中可见大量BMP2-DDPCs贴附于材料上,生长旺盛、伸展良好.结论 BMP2-DDPCs可在异种烧结骨表面及孔隙中生长、增殖,异种烧结骨可以作为牙组织工程的支架材料.%Objective To construct recombinant plasmid pEGFP - N1 - BMP2 and transfect dog dental pulp cells( DDPCs )with pEGFP - N1 - BMP2. BMP2 - DDPCs were seeded onto xenogeneic bone ceramice ( XBC ) scaffold as seed cells. To evaluate the feasibility of using XBC as scaffold for tissue engineered dentin, the biocompatibility between the seed cells( BMP2 - DDPCs ) and scaffold was detected by scanning electron microscopy and methyl thiazolyl tetrazolium( MTT ) analysis. Methods The full length dog BMP2 cDNA was linked into an eukaryotic expression vector pEGFP - N1. DDPCs were transfected with pEGFP - N1 - BMP2 by lipofectamine. The cell - scaffold compounds were made in vitro. The proliferative activity of BMP2 - DDPCs with XBC was detected by scanning electron microscopy( SEM ) and methyl thiazolyl tetrazolium( MTT ) analysis. Results pEGFP - N1 - BMP2 eukaryotic expression plasmid was constructed successfully. The constructed pEGFP - N1 - BMP2 could produce 4.7kb and 1.2kb fragments. The proliferative activity of BMP2 - DDPCs was not affected by XBC. BMP2 - DDPCs attached on the surface of XBC and stretched well. Conclusion BMP2 - DDPCs could attached on the surface of XBC ,grow and proliferate actively. XBC can meet the demand of dentin tissue engineering.
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