The Biologic Effect Of Ad-HIGF1 On The Endplate Cells

Object To try isolating and culturing rabbit endplate cells,and to observe the biological characteristics of the endplate cells.To learn the efficiency of the adenovirus vector carrying human insulin-like growth factor(hIGF1) transfecfing different generation rabbit cartilage endplate cells and the biologic effect of purpose gene on cell.To discuss the feasibility of applying Ad-hIGF-1 treating disc degeneration.Methods Cartilage endplate was isolated in vitro,and then was digested 30 minute with trypsin.We collected endplate cells after tissue being digested for different time with typeⅡcollagenase,and counted cell number and calculated cell survival rate by typan blue staining. Endplate cells were cultured monolayer through passage.The p1,p3,p6 cells were stained with ammonium methylbenzene blue,and we observed the different passage cells' shape.The gene sequence of Ad-hIGF-1 was tested and titre was measured using 50%tissue culture infectious dose.In order to study weather Ad-hIGF-1 could transfect into endplate cell,genomic DNA was extracted after cells being transfected 24 hours with Ad-hIGF-1,and then was tested using PCR, and the expression of hIGF-1 was measured by RT-PCR.After endplate cells being transfected with Ad-LacZ with 20,50,100,200 MOI respectively,we invested the optimum MOI applying marker gene Galacitiol nucleoside enzymes(LacZ).The p1,p3,p6 cells were transfected with Ad-hIGF-1,and then the gene expression of purpose gene hIGF1,aggrecan,collagenⅡwas semi-quantitatived by RT-PCR,and the capacity to proliferate was tested by MTT,and the secretion of hIGF-1 protein was tested by ELISA,and the secretion of aggrecan was tested by safranine O staining.Results We could harvest the largest cells number after tissue being digested 3 hours with typeⅡcollagenase,and the cells' survival rate was high.After 4 hours,the cell number decreased,and cells survival rate reduced.The first generation cartilage endplate cells performed triangular,polygonal,and stereo sense is strong.For the third cells,capacity for proliferation weakened,and appeared spindle shape.When cells were passaged to sixth passages,nucleus became vague,and cells appeared long spindle shape,and the refractive was weak,and the cells were changed into fibrosis.Being stained by ammonium methylbenzene blue,for the first generation cells,nucleus is dark blue,and the cytoplasm is light blue;for the third generation cells, cellular color is lighter than first generation cells,and for the p6 cells,staining color became apparently light compared with the p3 cells.The gene sequence of Ad-hIGF-1 is complete and the orientation is correct,judging from sequencing result.The titre of Ad-hIGF-1 was 2×10~9PFU/ml. Ad-hIGF-1 could transfect into endplate cells successfully,and cells could express purpose gene hIGF-1.When endplate cells were transfected with Ad-LacZ,transfection rate varied following multiplicity of infection within 100MOI range,and the transfection rate was both about 95%when multiplicity of infection were 100MOI and 200MOI respectively.When first generation cells were transfected with Ad-hIGF1,the gene expression of both hIGF1 and collage typeⅡincreased significantly compared with control group,and the expression of aggrecan didn't take place change(P＞0.05).When cells were passaged to third passage,the gene expression of hIGF1, aggrecan,collage typeⅡdidn't exist difference between cells which were passaged after being transfected at first generation and cells which were transfected after being passaged(P＞0.05). However,the gene expression increased compared with control group(P＜0.05).When cells were passaged to sixth passage,the gene expression of hIGF1,aggrecan,collage typeⅡdidn't exist difference between cells which were passaged after being transfected at first generation and cells which were transfected after being passaged(P＞0.05).However,the gene expression increased compared with control group(P＜0.05).At the same time,the capacity for proliferation of cells was enhanced while cells being transfected with Ad-hIGF-1,and the secretion of hIGF1 increased, and endplate cells could maintain its phenotype.Conclusion We cultured rabbit endplate cells in vitro successfully through isolation,culture, preliminary appraisal.The endplate cells degenerated gradually by monolayer culture.Therefore, the ceils could be studied as cartilage endplate models in vitro.Adenovirus vector carrying hIGF1 could transfect degenerated and normal endplate cells,and the transfection rate correlated positively with multiplicity of infection,and the transfection efficiency is the highest when multiplicity of infection is 100 MOI,and the adenovirus vector had not side-effect on cells.The rabbit endplate cells could express hIGF1 mediated by adenovirus vector,hIGF1 can promote cell expressing and secreting aggrecan,collage typeⅡ,and enhance cellular capability to proliferate,and maintain endplate cells phenotype,and the bioactivity could last long time.The biological effect of hIGF1 which was mediated by adenovirus vector was better when it was applied early disc degeneration than when it was applied latter disc degeneration.