重组2型腺相关病毒-人骨形成蛋白7转染犬髓核细胞及其对髓核细胞表型的影响

Effects of adeno-associated virus-2 mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells

中文关键词:  基因转染  人骨形成蛋白7  重组2型腺相关病毒  髓核细胞

英文关键词:Gene transfection  Bone morphogenetic protein-7  Recombinant adeno-associated virus type-2  Nucleus pulposus cell

中文摘要:

  【摘要】目的：观察重组2型腺相关病毒-人骨形成蛋白7（recombinant adeno-associated virus-2 expressing human bone morphogenetic protein-7，rAAV2-hBMP7）转染犬髓核细胞情况及其对细胞表型的影响。方法：用1岁龄Beagle犬L4/5椎间盘髓核进行髓核细胞体外培养，取第2代髓核细胞进行实验，实验组以 rAAV2-hBMP7（1×105v.g/细胞）转染髓核细胞，对照组以重组2型腺相关病毒-增强型绿色荧光蛋白（recombinant adeno-associated virus-2 expressing enhanced green fluorescent protein，rAAV2-EGFP）转染髓核细胞。在转染后4d、7d和14d以PCR检测髓核细胞中hBMP7的mRNA表达，在转染后7d和14d以Western blot法检测髓核细胞中hBMP7蛋白表达。在转染后4d、7d和14d采用RT-PCR法检测蛋白多糖、Ⅰ型胶原和Ⅱ型胶原的mRNA含量，采用二甲基蓝及ELISA法分别检测蛋白多糖、Ⅰ型胶原和Ⅱ型胶原的蛋白含量。结果：实验组髓核细胞表达hBMP7 mRNA及蛋白，并于转染后7d时表达量最高，而对照组无表达。实验组髓核细胞蛋白多糖mRNA及蛋白含量在转染后7d和14d时较4d时明显增高，14d时较7d时亦明显升高（P<0.05），对照组各时间点无统计学差异（P>0.05）；4d时实验组与对照组比较无明显差异，7d和14d时较对照组明显增加（P<0.05）。同组各时间点Ⅰ型胶原mRNA及蛋白含量均无统计学差异，两组各时间点间比较无统计学差异（P>0.05）。实验组髓核细胞Ⅱ型胶原mRNA及蛋白含量在转染后7d和14d时较4d时明显增高，14d时较7d亦明显升高（P<0.05），对照组各时间点无统计学差异（P>0.05）；实验组4d时与对照组比较无明显差异，7d和14d时较对照组明显增加（P<0.05）。结论：rAAV2-hBMP7转染犬髓核细胞后能表达hBMP7蛋白，并能提高其蛋白多糖及Ⅱ型胶原含量。

英文摘要:

  【Abstract】 Objective：To investigate the effects of recombinant adeno-associated virus-2 expressing human bone morphogenetic protein-7（rAAV2-hBMP7） on the phenotype of canine nucleus pulposus（NP）cells.Method：The NP cells were derived from lumbar 4-5 intervertebral disc of 1 year old beagle dog.The 2nd passage NP cells were cultured and transfected by rAAV2-hBMP7 at multiplicities of infection of 1×105 genomes per cell（experiment group），while the control group cells by recombinant adeno-associated virus-2 expressing enhanced green fluorescent protein（rAAV2-EGFP）.NP cells were assessed semi-qualitatively for BMP7 expression by RT-PCR at 4，7 and 14 days after transfection.BMP7 protein expressed by NP cells were assayed by Western blot at 7 and 14 days post-transfection.Aggrecan，type Ⅰ and type Ⅱ collagen secreted by NP cells were qualitatively assayed at 4，7 and 14 days after transfection in the transfection and control groups by dimethylmethylene blue（DMMB） and ELISA method.Result：The NP cell transfected by rAAV2-hBMP7 expressed human BMP7 mRNA and BMP7 protein.Their expression level reached peak at the 7th days.However，these proteins could not be detected in the control group at any time points.In experiment group，the accumulation of proteoglycans mRNA and protein at 7 and 14 days after transfection were remarkably higher than that of 4 days after transfection.Moreover，the accumulation of proteoglycans mRNA and protein at 14 days was higher than that of 7 days（P<0.05）.However，the accumulation of proteoglycans mRNA and protein showed no significant difference at any time points in the control group（P>0.05）.The accumulation of proteoglycans mRNA and protein in the experiment group was not different from that of the control group at 4 days，but higher than that of control group at 7 and 14 days（P<0.05）.The accumulation of type Ⅰ collagen mRNA and protein in each groups showed no difference at any time points.Moreover，there was no difference at each time point in both groups（P<0.05）.In the experiment group，the accumulation of Ⅱ collagen mRNA and protein at 7 and 14 days were remarkably higher than that at 4 days.Moreover，the accumulation of Ⅱ collagen mRNA and protein at 14 days post-transfection was higher than that at 7 days（P<0.05）.However，the accumulation of Ⅱ collagen mRNA and protein showed no significant difference at any time points in the control group（P>0.05）.The accumulation of Ⅱ collagen mRNA and protein in experiment group was not different from that of the control group at 4 days，but higher than that of the control group at 7 and 14 days（P<0.05）.Conclusion：The NP cells transfected by the rAAV2-hBMP7 can overexpress hBMP7.The rAAV2-hBMP7 is capable of promoting the expression of proteoglycans and type Ⅱ collagen of NP cells.