骨形成蛋白2基因转染对犬牙髓细胞生物学特性的影响

目的构建骨形态发生蛋白2（bone morphogenetic proteins 2,BMP2）绿色荧融合蛋白pEGFP-N1-BMP2真核表达质粒,然后再用其在体外转染犬牙髓细胞（DDPCs）形成BMP2-DDPCs细胞,观察BMP2基因转染对牙髓细胞生物学特性的影响。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DDPCs,检测BMP2-DDPCs碱性磷酸酶（ALP）活性,骨钙素（OC）产量和Ⅰ型胶原合成量等指标的测定,研究BMP2基因转染对牙髓细胞生物学特性的影响。结果成功构建pEGFP-N1-BMP2真核表达质粒,对构建的BMP2真核重组质粒用XhoI、HindIII进行双酶切,其产物进行琼脂糖凝胶电泳后,在1.2、4.7kb可见两条特异条带;并进行全基因序列测序,报告100%符合,证明pEGFP-N1-BMP2重组质粒构建成功。BMP2-DDPC中ALP活性,OC产量和Ⅰ型胶原合成量增加,与对照组DDPCs、N1-DDPCs相比差异有统计学意义（P〈0.05）,通过增强ALP、OC和Ⅰ型胶原等成骨标志物的表达,促进牙髓细胞向成牙本质细胞分化。结论 pEGFP-N1-BMP2真核表达质粒转染后的牙髓细胞,具有成牙本质细胞的特征。

The effect of gene transfection of bone morphogenetic proteins 2 on the cell biological characteristics of dog dental pulp cells in vitro

Objective To construct the eukaryotic expression plasmid of bone morphogenetic proteins 2(BMP2) and pEGFP-N1-BMP2,then,using the plasmid to transfect dog dental pulp cells(DDPCs) to form BMP2-DDPCs in vitro so as to observe the effect of BMP2 gene transfection on the cell biological characteristics of dog dental pulp cell.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 transfection.The activity of alkaline phosphatase(ALP) in BMP2-DDPCs was detected,and the contents of type Ⅰcollagen and osteocalcin(OC) in BMP2-DDPCs were measured.Results The pEGFP-N1-BMP2 eukaryotic expression plasmid was constructed successfully.The constructed pEGFP-N1-BMP2 could produce 4.7kb and 1.2kb fragments.The contents of alkaline phosphatase and OC were significantly increased in BMP2-DDPCs,as compared with those in control group(P<0.05).The results showed that the increase of the contents of ALP,OC and type Ⅰ collagen could promote dental pulp cells to differentiate toward odontoblast.Conclusion The DDPCs transfected with pEGFP-N1-BMP2 eukaryotic expression plasmid have the characteristics of odontoblast.