重组BMP7腺病毒的构建及转染兔髓核细胞的表达

Construction of BMP7 Recombinant Adenovirus and Expression of BMP7 in Transfected Rabbit Nucleus Pulposus Cells

目的构建重组BMP7腺病毒(Ad-BMP7),观察其转染兔髓核细胞后的表达。方法⑴真核表达载体pcDNA3.1(+)-BMP7经一系列特定的限制性内切酶酶切,重组了BMP7的腺病毒载体,并转染293细胞进行扩增、滴度测定。⑵取20只新西兰大白兔,15只作为实验组,5只作为实验对照组。实验组椎间盘注射Ad-BMP7,实验对照组注射携带绿色荧光蛋白(GFP)基因的腺病毒(Ad-GFP),另设空白对照组。分别于注射后1周、2周和4周各处死5只实验组兔子,注射后1周处死全部实验对照组兔子。RT-PCR和免疫组织化学染色检测BMP7的表达情况。结果⑴重组Ad-BMP7经限制性内切酶PI-SceⅠ和I-CeuⅠ双酶切获得BMP7基因片段,PCR扩增得到特异的扩增片段,表明重组的Ad-BMP7载体构建成功。⑵实验组的髓核细胞显示了BMP7染色阳性结果,1、2和4周组的阳性染色率差异无显著意义(P>0.05)。结论重组BMP7腺病毒的构建及其在兔髓核细胞的表达,为研究其对体内髓核细胞生物学行为的影响奠定了基础。

Objective To construct a recombinant adenovirus carrying human BMP7 gene which is transfected into rabbit nucleus pulposus cells and observe its expression. Methods （1） BMP7 adenovirus was recombined with a series of restrictive enzyme treatment of the pcDNA3.1 （ ＋ ）-BMP7, large-scale virus was prepared by using HEK 293 cells and viral titer was determined. （2） Twenty New Zealand white rabbits were chosen and 15 rabbits were in experimental group while 5 rabbits in the control group. Ad-BMP7, Ad-GFP carrying adenovirus 6 × 10^6 pfu was injected directly into rabbit nucleus pulposus of lumbar discs in experimental group and the control group respectively. Self control group remained for reference. The expression of BMP7 was detected by RT-PCR and immunohistochemistry （at 1st week, 2nd week and 4th week）. Results （1） BMP7 was able to be cut by the restrictive enzyme of PI-Sce Ⅰ and I-CeuⅠ from adenovirus BMP7 recombinant and BMP7 mRNA expression was observed by PCR, so it came true that we had constructed real adenovirus BMP7. （2） BMP7 gene was expressed effectively in the experimental group. There was no significant diffrence among the groups at the 1st week, the 2nd week and the 4th week （P 〉0.05）. Conclusion Construction of BMP7 adenovirus and its expression lays the basis for the further research in the bioactivity of the nucleus pulposus cells.