Effect Of Indian Hedgehog Protein In Cartilage Endplate Cells On Cartilage Endplate Degeneration

Objective:1.To observe the expression of IHH in different stages of human cartilage endplate(CEP);2.The significance of IHH expression on cartilage endplate degeneration was observed by culturing CEP chondrocytes in vitro.Methods: Part ?: The expression of Ihh protein in different stages of human cartilage endplate and its significance3.The cartilage endplate(CEP)specimens were collected from May 2018 to December 2018.The CEP-Modic degenerated tissues from patients with Modic ? or ? and normal tissues form lumbar vertebrae fractures patients were collected.4.The expression of Ihh,positive protective index of cartilage(Col ??Sox9)and index of cartilage hypertrophy(Runx2?MMP-13)in these specimens were detected and analyzed.The detection methods include Western-blot and real-time quantitative polymerase chain reaction(RT-qPCR).Part II: Experimental study on human cartilage endplate cells in vitro 1 Reagent intervention study:1.Chondrocytes isolated from CEP were cultured in vitro to P3 generation.They were divided into three groups: control,Ihh and anti-Ihh group according to extracellular intervention Ihh.2.PBS,Ihh recombinant protein solution and anti-Ihh protein solution were added to the culture medium separately,and the chondrocytes were cultured for 3-5 days.The expression of Ihh and related indexes were detected by Western-blot and RT-qPCR.Cells coverslips were stained by using fluorescent immunocytochemistry and fluorescent in situ hybridization.Then data were analyzed by using protein gray value,mRNA expression level and mean optical density value analysis.2 Plasmid transfection study:1.Chondrocytes isolated from CEP were cultured in vitro to P3 generation.They were divided into three groups: empty vector as control,Ihh over-expression group and Ihh knockdown group according to intracellular intervention Ihh.2.Lipo2000 carrying Ihh mRNA,Ihh siRNA and empty vector were used to transfer chondrocytes separately,and the 3 groups chondrocytes were cultured for 3-5 days.The expression of Ihh and related indexes were detected by Western-blot and RT-qPCR.Cells coverslips were stained by using fluorescent immunocytochemistry and fluorescent in situ hybridization.Then data were analyzed by using protein gray value,mRNA expression level and mean optical density value analysis.Results:Part ?: The expression of Ihh protein and mRNA in the CEP tissues of patients with Modic I and II were expressed more than normal tissues,and the expression was increased in Modic I and II laminitis tissues.The positive protective index of cartilage(Col II,Sox9)expression decreased,while the expression of index of cartilage hypertrophy(Runx2?MMP-13)increased.In the RT-qPCR results,there was no difference in expression between Modic I type and type II cartilage tissues.Part II:1.The primary CEP tissue chondrocytes were round,and the cells were short shuttlelike after adhering to the wall.The P3 generation adherent cells were mostly short shuttlelike and less triangular,furthermore,the cells were small volume and excellent in refractive index.After the addition of the reagents,the Ihh group showed more longer shuttle-like cells than the control and anti-Ihh groups under the same light microscope field(40×).Compared among 3 groups,Ihh and Runx2,MMP-13 mRNA expression levels were relatively increased,Sox9 mRNA expression(p<0.05)and Col II mRNA were reduced,however,reduction of Col II mRNA was not statistically significant(p>0.05)in the Ihh group.The relative expression of Col II mRNA was significantly increased in the anti-Ihh group,and the relative expression of Ihh,Runx2 and MMP-13 mRNA was decreased(p<0.05).But the reduction of Sox9 mRNA expression was not statistically significant(p>0.05).Western-blot gray value analysis indicated that Ihh and MMP-13 protein in Ihh group was stronger,Col II was weaker,in addition anti-Ihh group was opposite(p<0.05).Compared with control group,fluorescent immunocytochemistry and fluorescent in situ hybridization also showed corresponding results.The mean optical density value of the staining results showed that Ihh and MMP-13 were more in the Ihh group and less Col II(p<0.05),while the anti-Ihh group was opposite(p<0.05).2.In the Ihh over-expression group,the most chondrocytes were dedifferentiated and even some cells died.In the control group,the chondrocytes were polyhedral and short shuttle-like form.The chondrocytes were short shuttle-like and small size in the knockdown group.They were studied under the same light microscope field(40×).Compared among 3 groups,Ihh and Runx2,MMP-13 mRNA expression levels were relatively increased,Sox9 mRNA expression(p<0.05)and Col II mRNA were reduced,however,reduction of Col II mRNA was not statistically significant(p>0.05)in the Ihh over-expression group.The relative expression of Col II mRNA was significantly increased in the Ihh knockdown group,and the relative expression of Ihh,Runx2 and MMP-13 mRNA was decreased(p<0.05).But the reduction of Sox9 mRNA expression was not statistically significant(p>0.05).Westernblot gray value analysis indicated that Ihh and MMP-13 protein in the Ihh over-expression group was stronger,Col II was weaker,in addition Ihh knockdown group was opposite(p<0.05).Compared with control group,fluorescent immunocytochemistry and fluorescent in situ hybridization also showed corresponding results.The mean optical density value of the staining results showed that Ihh and MMP-13 were more in the Ihh over-expression group and less Col II(p<0.05),while the Ihh knockdown group was opposite(p<0.05).Conclusion:1.The expression of IHH protein in Modic ? and ? CEP tissues was increased,and decreased in normal tissues;2.Increased Ihh expression in cartilage endplate cells could lead to degeneration of c chondrocyte.Decreased Ihh expression could maintain chondrocyte phenotype and delay degeneration.