GDF-5基因体外转染骨髓间充质干细胞移植与GAM体内转染对椎间盘退行性变作用的动物实验研究

Comparison of the effect of in vitro GDF-5-transfected BMSC transplantation and in vivo GAM transfection on the repair of IDD in rabbit

目的通过对比体外生长分化因子5（GDF-5）转染并移植和基因活化基质（GAM）体内转染对椎间盘退行性变的修复作用，评价这两种方法的差异，以期为临床应用提供依据。方法日本大耳白兔32只，体质量约1.5～2.0kg．雌雄不限。取兔骨髓，分离骨髓间充质干细胞（BMSC），用脂质体介导的pcDNA3.1（＋）／GDF-5基因重组质粒转染BMSC。将兔随机分为A组（正常组）、B组（转染细胞组）、C组（GAM组）、D组（对照组）。B、C、D组从L1-2、L2-3、L3-4、L4-5抽吸约湿质量5mg的髓核组织，制作椎间盘退行性变动物模型；B组植入已转染GDF-5的BMSC，C组植入GDF-5GAM，D组植入空白培养液．术后2个月将包括软骨终板在内的各组完整的椎间盘取出，采用间苯三酚法测量椎间盘髓核中蛋白多糖含量．采用流式细胞仪检测分析髓核细胞凋亡率。结果转染48h后BMSC生长良好，体积略增大。经测定pcDNA3.1（＋）／GDF-5基因转染成功后髓核内蛋白多糖含量，B组（0.2169±0.0264）与A组（0.2401±0.0300）间差异无统计学意义（P=0.149）；其余组（C组、D组分别为0.1667±0.0278、0.1153±0.0337）两两比较，差异都有显著的统计学意义（P〈0.01）。髓核细胞凋亡率，A组（24.55％±2.681％）与B组（30.980％±2.462％）间差异具有统计学意义（P=0.012），其余各组（C组、D组分别为38.220％±2.524％、57.530％±2.349％）两两相比,差异都有显著统计学意义（P〈0.01）。结论相对于利用GAM进行体内转染,体外转染BMSC然后再移植人体内的方法修复椎间盘退行性变的效率更高。

Objective To compare the repairment effect on disc degeneration of in vitro GDF-5- transfected BMSC transplantation and in vivo GAM transfection, and provide experimental fundamentals for their clinical application. Methods BMSC isolated from Japanese white rabbit were transfected with peDNA3.1 （＋）/GDF-5 using lipofectamine as transfection reagent. The 32 Japanese white rabbits were randomly divided into 4 groups（8 rabbits each）, with group A as normal group, group B as transfected cell group, group C as GAM group, and group D as control group. IDD models were established by taken out about 5 mg nucleus pulposus tissue from L1-2, L2-3, L3-4、 L4-5 of group B, C and D, and thereafter, different materials were injected into the defected part（GDF-5-BMSC to group B, GDF-5-GAM to group C and cultured fluid to group D. Two-month after the operation, the complete set of discs included cartilage endplate was taken out and observed with light microscope and TEM. The content of nucleus pulposus proteoglycan was measured by the phloroglucinol method and the apoptosis of nucleus pulposus cells was analyzed with flow cytometer. Results The ceils grew well with a little larger volume 48-hour post- transfection. Two months after the treatment, annular fibrosus ruptured partially when observed under light microscope, and apoptotic body, vacuolus in cytoplasm and incomplete chondrosome were observed under TEM. RT-PCR analysis demonstrated that group B and C were successfully transfected. No significant difference in nucleus pulposus proteoglycan content was found between group B（0.216 9±0.026 4） and group A（0.240 1 ± 0.030 0）（P = 0.149） while the difference between group C and group D was statistically significant（P 〈 0.01）. The difference in apoptosis rate of nucleus pulposus cells between group A（24.550 % ± 2.681%） and B（30.980 % ±2.462 %） was significant（P= 0.012）, and the difference between group C and group D was statistical signifieant（P 〈 0.01）. Conclusion In vitro GDF-5-transfeeted BMSC transplantation can repair the IDD more efficiently than in vivo GAM transfection.