联合应用BMP-2及细胞分化抑制因子1基因延缓兔腰椎间盘退变的动物实验研究

Transfection of lentivirus-bone morphogenetic protein 2 and lentivirus-inhibitor of differentiation 1 into nucleus pulposus for delaying intervertebral disc degeneration in an in vivo rabbit model

目的研究兔腰椎间盘髓核内注射慢病毒携带的BMP-2和细胞分化抑制因子1(inhibitor of differentiation 1,Id1)基因能否有效改善椎间盘退变进程。方法取4月龄新西兰兔32只,体质量2.0~2.5 kg;以L3、4、L4、5、L5、6为目标椎间盘,经腹细针穿刺法制造椎间盘退变模型。将30只造模成功的实验动物随机分为5组,每组6只;造模后4周于各组目标椎间盘中分别注射Lv-BMP-2病毒(A组)、Lv-BMP-2+Lv-Id1病毒(B组)、LvId1病毒(C组)、Lv-绿色荧光蛋白空载体病毒(D组)及PBS(E组)。于注射后2、4、8周各组分别随机取2只动物行T2-mapping MRI检查获得确切的T2弛豫时间(T2值);之后处死动物取椎间盘组织,应用实时荧光定量PCR、ELISA法检测Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量。结果 T2-mapping MRI检查示,注射后0、2周各组间T2值比较差异均无统计学意义(P>0.05);注射后4周,A、B组T2值显著高于C、D、E组(P<0.05),A、B组间比较差异无统计学意义(P>0.05);注射后8周,B组T2值显著高于其余各组(P<0.05)。实时荧光定量PCR及ELISA检测示,注射后2、4、8周B组Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量均显著高于其余各组(P<0.05)。结论联合应用BMP-2和Id1基因在体内实验中能有效延缓兔腰椎间盘退变的进程。

Objective To investigate if the course ofintervertebral disc degeneration （IDD） is delayed by injecting lentivirus （Lv） vector carrying bone morphogenetic protein 2 （BMP-2） and inhibitor of differentiation 1 （Id 1） genes directly into the nucleus pulposus. Methods Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L3,4, L4, 5, and L5, 6 discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 （group A）, with Lv-BMP-2 and Lv-Idl （group B）, with Lv-Id1 （group C）, with Lv-green fluorescent protein （group D）, and with PBS （group E）. At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. Results T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection （P〉0.05）. The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection （P〈0.05）, but no significant differencewas observed between group A and group B （P〉0.05）. The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection （P〈0.05）. The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection （P〈0.05）. Conclusion Combined application of Lv-BMP-2 and Lv-Idl can delay IDD changes in rabbit IDD models.