hIGF1基因转染促进退变椎间盘Ⅱ型胶原表达.docVIP

hIGF-1基因转染促进退变椎间盘Ⅱ型胶原的表达 作者：黄宗强，刘尚礼，郑召民 【摘要】 ［目的］探讨人胰岛素样生长因子（human insulin-like growth factor-1,hIGF-1)基因在退变椎间盘中的表达及对椎间盘中Ⅱ型胶原表达的影响。［方法］参考文献〔1〕制备出腰椎间盘退变（intervertebral disk degeneration,IVDD)动物模型24只，随机分为携带hIGF-1基因重组腺病毒（Ad/CMV-hIGF-1)、hIGF-1生长因子及PBS组。L4、5、L5、6椎间盘中分别注射25 μl第2代Ad/CMV-hIGF-1(8×108 PFU)、hIGF-1生长因子（100 μg/L)、PBS。注射后1、2、4、8周，Western blot检测hIGF-1蛋白表达；半定量RT-PCR检测Ⅱ型胶原mRNA的表达。［结果］hIGF-1蛋白带出现在7 540.00 u。Ad/CMV-hIGF-1组hIGF-1蛋白表达持续达4周以上，hIGF-1组表达持续约2周；PBS注射组无hIGF-1蛋白表达。Ⅱ型胶原电泳条带出现在200～300 bp；在注射后1～4周，Ad/CMV-hIGF-1组内Ⅱ型胶原mRNA相对表达量进行性增加，8周轻度下降，4个时期比较（F=12.18，P＜0.001)；注射后1～8周，hIGF-1注射组、PBS注射组Ⅱ型胶原mRNA相对表达量进行性下降（F=27.38、21.56，P＜0.001)。相同时间点3组比较，Ⅱ型胶原mRNA相对表达量Ad/CMV-hIGF-1组最高，PBS组最低（F=27.31～39.85，P＜0.001）。［结论］hIGF-1基因在退变椎间盘中的表达能够促进合成Ⅱ型胶原。 【关键词】 椎间盘； 退变；表达； hIGF-1 Abstract［Objective］To study the role of hIGF-1 gene on collagen type Ⅱ expression of degenerative intervertebral disk.［Method］Twenty-four male New-Zealand rabbits intervertebral disk degenerontion (IVDD) models were done according to reference and randomly divided into Ad-CMV-hIGF-1,hIGF-1 growth factor and PBS group.Twenty five microlitre the second generation Ad/CMV-hIGF-1(T=80×109 PFU/L),hIGF-1 growth factor(100 μg/L)and PBS were respectively injected into L4、5,L5、6 intervertebral disk under fluoroscopic guidance.One,two,four and eight weeks post-operation,rabbits were sacrificed,intervertebral disk samples were harvested.Total proteins of equal mass intervertebral disks were extracted,isolated in SDS-Polyacrylamide gel electrophoresis(SDS)and transferred to polyvinylidene difluoride(PVDF)Millipore.The hIGF-1 growth factor expression were indentified with Western blot.Collagen type Ⅱ gene fragments were amplified with RT-PCR,and relative expression was done with GAPDH as intern control.［Result］The hIGF-1 interest protein existed at 7.6 Kilo-Dalton.At one week after injection,its expression quantities were almost equal between Ad/CMV-hIGF-1 and hIGF-1 growth factor group.At two week after injection,it obviou