GDF-5基因重组腺病毒修复人退变髓核细胞的实验研究

An experimental study of adenovirus-mediated GDF-5 restoring the degenerative nucleus pulposus cells of human

目的探讨腺病毒介导的GDF-5基因对人退变髓核细胞外基质表达的影响,探求一种基因治疗新途径。方法利用腺病毒载体将GDF-5基因导入退变髓核细胞,设置为空白对照组(Control组)、阴性对照组(GFP组)、实验组(GDF-5组)3组,并分为3、7、14、21天四个观察时间点,分别检测3组髓核细胞sGAG、Hyp的含量,免疫染色、Safranine-O染色观察细胞外基质合成情况,Real-timePCR检测Aggrecan、CollagenⅡmRNA表达水平。结果GDF-5组髓核细胞sGAG/DNA、Hyp/DNA均在14、21天较Control组和GFP组明显增加,差异有统计学意义(P<0.05),而Control组与GFP组之间任意观察时间点比较差异均无统计学意义(P>0.05);免疫染色、Safranine-O染色结果显示GDF-5组髓核细胞的蛋白多糖、Ⅱ型胶原、蛋白聚糖在14、21天均呈强阳性染色,Control组和GFP组呈弱阳性染色。Real-timePCR结果显示GDF-5组Aggrecan、CollagenⅡmRNA的表达在14、21天较Control组和GFP组明显增加,差异有统计学意义(P<0.05)。结论腺病毒介导的GDF-5能明显促进人退变髓核细胞Aggrecan、CollagenⅡmRNA的表达,并增加蛋白多糖、Ⅱ型胶原的合成,对细胞外基质具有明确的修复作用,是一种有效的基因治疗新方法。

Objective To investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells,and explore a candidate gene therapy method for intervertebral disc degeneration (IDD).Methods NP cells were isolated,cultured,and then infected by the optimal multiplicity of infection (MOI) of Ad-GDF-5 were set up as experimental group (GDF-5 group),and compared with blank control group (Control group) and Ad-GFP control group (GFP group).The extracellular matrix expressions of NP cells were evaluated at 3,7,14 or 21days.The contents of sulfated glycosaminoglycan (sGAG) and hydroxyproline (Hyp) in NP cells were measured.The expression of aggrecan,type-Ⅱ collagen and proteoglycan were observed respectively by immunofluorescent staining,immunohistochemical staining and Safranine-O staining.The transcriptions of aggrecan and collagen Ⅱ mRNA were analyzed by real-time quantitative polymerase chain reaction (Real time-PCR).Results The contents of sGAG and Hyp in NP cells gradually increased following cells culture in all three groups.The GDF-5 group exhibited a significantly higher sGAG/DNA than GFP and Control groups on 14 or 21 days ( P <0.05),and showed a significantly greater Hyp/DNA than GFP and Control groups on 7,14 or 21 days ( P <0.05),whereas GFP group did not show a greater effect than Control group ( P >0.05).The NP cells were stained for aggrecan (Immunofluorescent staining),type-Ⅱ collagen (Immunohistochemical staining) and proteoglycan (Safranine-O staining).The NP cells infected by GDF-5 exhibited robust staining on 14 or 21 days,and weakly staining intensity either GFP or Control group.Levels of RNA transcripts for aggrecan and collagen Ⅱ expression patterns were determined in NP cells using real-time PCR.Compared with GFP or Control groups,GDF-5-treated group had a significantly up-regulated aggrecan and collagen Ⅱ expression levels on 14 or 21 days point ( P <0.05),whereas GFP group did not demonstrate a significant enhancement than Control group ( P >0.05).Conclusion Ad-GDF-5 could obviously up-regulate aggrecan and collagen Ⅱ mRNA transcription in NP cells and increase the synthesis of proteoglycan and type-Ⅱ collagen.Ad-GDF-5 could restore the extracellular matrix of degenerated NP cells and could be a potential candidate method of gene therapy for IDD.