An Experimental Study Of Adenovirus Mediated Growth And Differentiation Facotr-5 Promoting The Extracellular Matrix Expression In Nucleus Pulposus Cells Of Degenerative Intervertebral Disc

Objective: To construct recombinant adenovirus vector carrying thehuman growth and differentiation factor-5 (GDF-5) gene and investigate thebiological effects of adenovirus mediated GDF-5 on extracellular matrixexpression in nucleus pulposus (NP) cells of human degenerativeintervertebral disc and explore a candidate method of gene therapy forintervertebral disc degeneration (IDD).Methods: (1) Recombinant adenovirus vector GDF-5 was constructedusing the AdEasy-1 adenovirus vector system. The adenovirus vectors weretransfect to HEK 293 cells to package, amplify and purify and identified byrestriction enzyme digestion, gene sequencing and western blotting. The virustiter was determined by TCID50. (2) NP tissues of human degenerativeintervertebral disc were collected and NP cells were isolated and cultured invitro. The NP cells infected by the optimal multiplicity of infection (MOI) ofAd-GDF-5 were set up as experimental group (GDF-5 group), and comparedwith Ad-GFP control group (GFP group) and blank control group (Controlgroup). The extracellular matrix expressions were evaluated at 3, 7, 14 or21days. (3) The contents of sulfated glycosaminoglycan (sGAG) in NP cellswere measured spectrophotometrically after incubation with 1,9-dimethylmethylene blue-chloride (DMMB) dye and normalized to total DNA, as measured fluorometrically using the bisbenzimide Hoechst 33258DNA quantitation kit. (4) The contents of hydroxyproline (HYP) amino acidin NP cells were detected by chloramines T staining. (5) The expression ofproteoglycan and collagen type II were observed by immunofluorescent orimmunohistochemical staining. (6) The sulfated glycosaminoglycan (sGAG)was also evaluated by Safranine-O staining. (7) The transcriptions ofaggrecan and collagenⅡmRNA were analyzed by real-time quantitativepolymerase chain reaction.Results: (1) Recombinant adenovirus vector GDF-5 was successfullyconstructed and confirmed by restriction enzyme digestion, gene sequencingand western blotting. The virus titer was 5.6×109PFU/ml. (2) Contents ofsGAG gradually increased following cells culture in all three groups, andGDF-5 group exhibited a significantly greater extent than either Control orGFP group on 14 days (P<0.05) and particularly on 21 days (P<0.01).Nevertheless, no significant difference was observed between Control groupand GFP group. (3) There are no significant differences of contents ofhydroxyproline amino acid in three groups on 3 days (P>0.05), and GDF-5group showed a significantly greater extent than either Control or GFP groupon 7days (P<0.05). Especially after 14 or 21days culture, significantdifferences were clearly observed in GDF-5 group compared with control orGFP group (P<0.01), whereas no significant difference between control groupand GFP group at this time point (P>0.05). (4) The NP cells were stained forProteoglycan (immunofluorescent staining), collagen type II(immunohistochemical staining) and sGAG (Safranine-O staining). All NPcells demonstrated weakly staining intensity on 3 days or 7days. But NP cellsinfected by GDF-5 demonstrated robust staining on 14 or 21 days point and the staining intensity was higher than either control or GFP group. (5) After14 days in culture, mRNA transcription of aggrecan or collagenⅡup-regulated in all three groups, but GDF-5 group provided a significantenhancement compared to Control or GFP group (P<0.05). On 21days, bothaggrecan and collagenⅡmRNA levels in GDF-5 group were significantlyhigher than any other groups (P<0.01). However, no significant differenceswere observed between control group and GFP group.Conclusion: (1) Recombinant adenovirus vector carrying the humanGDF-5 gene was successfully constructed using the AdEasy-1 adenovirusvector system. (2) The Ad-GDF-5 with high titer after amplified in HEK 293cells could produce mature GDF-5 proteins. (3) The NP cells of humandegenerative intervertebral disc were isolated and cultured in vitro and wereoptional target cells for GDF-5 gene therapy mediated by adenovirus. (4)Ad-GDF-5 could significantly up-regulate aggrecan and collagenⅡmRNAtranscription in NP cells and increase the synthesis of proteoglycan andcollagen type II. (5) Ad-GDF-5 could promote the extracellular matrixexpression in NP cells and restore the functions of degenerative intervertebraldisc and could be a potential candidate treatment for intervertebral discdegeneration.