人骨形态发生蛋白2重组腺病毒载体的构建及其生物学活性检测

Construction of recombinant adenoviral vector encoding human BMP-2 and detection of its biological activities

目的构建人骨形态发生蛋白2(hBMP-2)重组腺病毒载体(Ad-hBMP-2),并检测其转染后的骨髓基质细胞hBMP-2表达以及成骨能力的改变。方法采用Cre-lox重组方法构建含有hBMP-2基因的复制缺陷型重组腺病毒,并利用原位杂交和免疫组化手段观察转染后的骨髓基质细胞hBMP-2mRNA和蛋白表达情况;通过Ⅰ型胶原原位杂交、碱性磷酸酶测定、透射电镜观察以及裸鼠肌袋试验评价Ad-hBMP-2转染对兔骨髓基质细胞成骨能力的影响。结果病毒培养上清液的聚合酶链式反应证实构建的重组腺病毒含有Ad-hBMP-2基因,重组腺病毒滴度达3·8×1010pfu/L;感染增殖率为100的Ad-hBMP-2转染兔骨髓基质细胞24h和48h后即可分别检测到显著的hBMP-2mRNA和蛋白表达,转染效率几乎达100%;Ad-hBMP-2转染可以明显刺激兔骨髓基质细胞Ⅰ型胶原mRNA表达、碱性磷酸酶分泌、细胞内蛋白质合成以及异位骨形成等。结论Ad-hBMP-2具有高效转染能力,并能够诱导骨髓基质细胞向成骨细胞转化,进而促进骨形成。

Objective To construct an adenoviral vector containing human BMP-2 cDNA（Ad-hBMP-2）, and to examine the expression of human BMP-2 gene and osteogenic abilities in transfected BMSCs. Methods Ad-BMP-2 was generated by Cre-lox recombination; the expression of hBMP-2 mRNA and protein was detected by in situ hybridization and immunohistochemistry in transfected BMSCs; the osteogenic activities of transfected BMSCs were observed by expression of type Ⅰ collagen mRNA, determination of ALP activity, observation of ultrastructural features and investigation of ectopic bone formation. Results Ad-hBMP-2 was determinated by PCR reaction, the titer of Ad- hBMP-2 was 3.8 × 10^10 pfu/L; Almost 100% of BMSCs showed positive hBMP-2 mRNA expression after transfected for 24 h, and positive hBMP-2 protein expression after transfected for 48 h by Ad-hBMP-2 at an MOi of 100 ; in BM- SCs group transfected by Ad-hBMP-2, positive or strong positive expression of type Ⅰ collagen mRNA, increased ALP activity, dilated rough endoplasmic reticulum with abundant proteinoid substance and ectopic bone formation could be observated. Conclusions Ad-hBMP-2 has a high transfection efficiency, may induce the differentiation of BMSCs to osteoblasts, and further enhance bone formation.