Effect Of ID1 Gene On BMP-2 Maintaining The Cartilage Phenotype Of Rabbit Intervertebral Disc Cells

Objective:To construct lentiviral vector for overexpression and RNA interference of Idl gene, the nucleus pulposus cells of rabbit intervertebral disc is intended to be the research object, observe the change of nucleus pulposus cells chondroblast features with the induction of BMP-2 when the expression level of Idl gene is adjusted, study the effect of Id1 on BMP-2 maintaining the cartilage phenotype of intervertebral disc cells.Methods:(1) To design the sequence for overexpression and RNA interference of rabbit Id1 gene, linked into lentivirus vector by using gene recombination technology, then infect the constructed lentiviral vectors into 293T cells, complete the gene sequencing and determin the titer after infection. (2)Inject the two kinds of recombinant lentiviruses into nucleus pulposus cells, assay the expression of Idl mRNA and protein by real-time quantitative PCR and western blotting. (3)Transfect the nucleus pulposus cells by AdBMP2, then using the two kinds of lentivirus transfect the cells again, detect the ability and trend of expressing cartilage phenotype molecular by QRT-PCR and ELISA.Results:(1) We constructed the lentivirus vector of Idl for overexpression and RNA interference successfully, the sequencing consistent with Idl gene sequence after confirmation.(2)The expression quantity of Id1 gene and protein in nucleus pulposus cells were changed after lentiviral transfection:compared with the normal cell control group and negative control group, the expression quantity of overexpression group were increased, while the group of RNAi decreased, thus conform to the expected results.(3)The boost of Idl expression level can promote the mRNA expression of collagen Ⅱ and agreecan. (4)After infecting the cells by AdBMP2, the boost of Idl expression level can promote the mRNA expression of collagen Ⅱ (0.590±0.072) and agreecan (0.351±0.066) more significantly while the decrease of Id1 expression can inhibit the expression of collagen Ⅱ and agreecan. (col Ⅱ.0.244±0.060, ACAN:0.168±0.034).Conclusion:Id1 can promote the expression of cartilage feature molecule, the change of Id1 gene inside the nucleus pulposus cells can significantly affect the secretion of cartilage feature molecule which induced by BMP-2, thus it can be seen that Id1 is the key factor in BMP-2 maintaining the cartilage phenotype of rabbit nucleus pulposus cells.