rAAV2-CTGF和TIMP1双基因联合转染恒河猴退变腰椎间盘的生物学效应

目的 研究腺相关病毒(adeno-associated virus 2,AAV2)介导的结缔组织生长因子(con-nective tissue growth factor,CTGF)和基质金属蛋白酶组织抑制因子1(tissue inhibitor of metalloproteinas-es 1,TIMPl)双基因体内转染退变的恒河猴腰椎间盘的生物学效应.方法 恒河猴9只,雌性4只,雄性5只;年龄4～7岁,体重4.5～7 kg.CT引导下经皮穿刺纤维环诱导恒河猴腰椎间盘退变,微创经皮注入携带CTGF和TIMP1双基因腺相关病毒颗粒.应用RT-PCR测定细胞因子的表达,RT-PCR、35S整合法观察双基因联合转染对退变椎间盘的生物学作用.结果 双基因转染后8、16、24周时CTGF mRNA表达量分别为PBS对照组的10.02、2.39、0.91倍;TIMP1 mRNA表达量分别为PBS对照组的6.08、3.81、2.67倍;Ⅱ型胶mRNA表达量分别为PBS对照组的145.51%、174.72%、113.73%;蛋白多糖mRNA表达量分别为PBS对照组的461.19%、191.46%、301.39%;蛋白多糖合成效率为分别PBS对照组的455.06%、285.97%、165.58%.结论 CTGF和TIMP1双基因联合转染后可以在体内较长期表达,并能促进体内蛋白多糖和Ⅱ型胶原的合成,延缓椎间盘的退变.

Biological effects of CTGF combined with TIMP1 mediated by adeno associated virus to degenerated lumbar intervertebral discs in rhesus monkey

Objective To study the biological effect of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMPI) mediated by adeno-associated virus 2 (AAV2) to rhesus monkey lumbar intervertebral discs in vivo. Methods To establish a novel model of lumbar disc degeneration in the rhesus monkey using percutaneous needle puncture guided by CT. The rAAV2-CTGF-IRES-T1MPI was injected percutaneously with minimal invasive technique. The mRNA expression of CTGF and TIMP1 was determined through RT-PCR. The biological effect of rAAV2-CTGF-IRES-TIMP1 was examined by RT-PCR, ~(35)S incorporation assay. Results At the 8th, 16th, 24th week after gene transfection, the expression of CTGF mRNA were 10.02, 2.39, 0.91 times respectively compared with the PBS control group; TIMP1 mRNA expression were 6.08, 3.81, 2.67 times respectively compared with the PBS control group; collagen type Ⅱ mRNA expression for the PBS control group were 145.51%, 174.72%, 113.73% respectively; proteoglycan mRNA expression were 461.19%, 191.46%, 301.39% respectively compared with the PBS contrel group; the efficiency of proteoglycan synthesis were 455.06%, 285.97%, 165.58% respectively compared with PBS control group. Conclusion The longer-term expression of CTGF and TIMP1 genes mediated by adeno-associated virus can achieve in vivo after transferring into the intervertebral disc, and also can increase the proteoglycan and collagen type Ⅱ synthesis, which delay disc degeneration.