生长分化因子-5(GDF-5)促进人退行性变髓核细胞外基质表达情况的研究（英文）

Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells

目的：研究腺病毒介导的GDF-5对人退行性变椎间盘髓核细胞生长和细胞外基质表达的影响，探索椎间盘退行性变基因治疗的新途径。创新点：首次验证了GDF．5对人退行变的髓核细胞的生长和细胞外基质的分泌均具有明显的促进作用，为椎间盘退行性变疾病早期基因治疗提供了新的途径。方法：利用腺病毒介导的GDF．5转染人退变的髓核细胞，设空白对照组、阴性对照组（绿色荧光蛋白（GFP）组）和实验组（GDF-5组）三个组，3、7、14和21天四个时问点。在预定的时间点，通过蛋白质免疫印迹（Westernblotting）、番红-O染色、免疫组化染色、酶联免疫法定量检测（ELISA）、逆转录聚合酶链式反应（RT-PCR）和细胞外基质的定量分析等手段，验证GDF-5对退变髓核细胞外基质表达的影响，同时对GDF．5对髓核细胞生长情况的促进作用进行了表征。结论：Ad—GDF-5能成功转染人退变的髓核细胞（图1），能有效地促进人退变髓核细胞细胞外基质Aggreean和Ⅱ型胶原（CollagenⅡ）分泌（图4-7），RT-PCR的结果表明Aggrecan和CollagenII基因表达也得到了明显的增强（图8）。同时GDF-5对人退变的髓核细胞的生长也有一定的促进作用（图9）。因此，Ad-GDF-5是椎间盘退行性变早期基因治疗的新途径。

Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, 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： Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan （sGAG）, deoxyribonucleic acid （DNA） and hydroxyproline （Hyp）, synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results： The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium （BM） or Ad-green fluorescent protein （GFP） groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions： The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP ceils.