Effect And Mechanism Of BMP9 On Maintaining Cartilage-like Properties Of Human Degenerative Nucleus Pulposus Cells

Intervertebral disc degeneration(IDD)is one of the important causes of low back pain and other symptoms.With the advancement of medicine,the prolongation of life expectancy and China enter into an aging society gradually,the incidence of IDD has increased significantly,seriously affecting the quality of life of patients,and also bringing heavy medical burden to society.The degeneration of intervertebral disc begins with the degeneration of nucleus pulposus(NP).The degeneration of nucleus pulposus is manifested in the gradual decrease of the main components of extracellular matrix(ECM),aggrecan and type II collagen(Col2a1),which make nucleus pulposus lose its elastic modulus and become fibrotic.At present,for the degeneration of nucleus pulposus tissue,whether surgical or conservative treatment,there are some shortcomings.Therefore,it is our main goal,through the transduction of exogenous substances,to delay or even reverse the degeneration of nucleus pulposus tissue.In the previous work of our group,BMP9 was transduced into the immortalized rat intervertebral disc cell line.It was found that the expression of Aggrecan and Col2a1 increased significantly.Therefore,we speculate whether BMP9 has similar effects on human degenerative nucleus pulposus cells(NPCs).This topic will be elaborated from the following four aspects: 1.Extraction,culture and identification of human degenerative nucleus pulposus cells;2.Effect of BMP9 on cell cycle,proliferation and ECM of human degenerative NPCs;3.The relationship between BMP9 and Notch signaling pathway and the regulation of Notch signaling in NPCs by BMP9;4.Regulation of ECM by dnNotch1,DLL1 and Jagged1 in NPCs.PART1.EXTRACTION,CULTURE AND IDENTIFICATION OF HUMAN DEGENERATIVE NUCLEUS PULPOSUS CELLSObjective To explore a complete of methods for extracting and culturing human degenerative nucleus pulposus cells and to identify them.Methods The specimens of lumbar intervertebral disc tissue were collected from the patients,who were take operation in the orthopaedic department of the First Affiliated Hospital of Chongqing Medical University.The specimens were immediately transferred to the cell operating table.After washing and careful separation,the nucleus pulposus tissue was obtained.After digested by enzyme sequentially,the cell suspension was obtained,and then inoculated into T25 culture flask and cultured in incubator.Nucleus pulposus cells(NPCs)were identified by immunofluorescence.Results The initial adherence time of primary nucleus pulposus cells was at least 3 days,the confluence rate to 90% was about 13-15 days,and the culture conditions of nucleus pulposus cells were relatively high.The presence of aggrecan and Col2a1 in the cells was detected by immunofluorescence,which proved that the cells were nucleus pulposus cells.Conclusion Pure nucleus pulposus cells can be extracted and culturedby enzymatic sequential digestion.PART2.THE EFFECT OF BMP9 ON CELL CYCLE, PROLIFERATION AND ECM OF HUMAN DEGENERATIVE NPCSObjective To investigate the effect of BMP9 on cell cycle,proliferation and ECM of human degenerative NPCs.Methods P1 nucleus pulposus cells were infected with Ad-GFP and Ad-BMP9.BMP9 was used as experimental group and GFP as control group.RT-PCR was used to verify the successful expression of BMP9.CCK-8 was used to detect the effect of BMP9 on the proliferation of NPCs.Flow cytometry was used to detect the effect of BMP9 on NPCs cycle.RT-PCR,Western blot and immunofluorescence were used to detect the expression of aggrecan and Col2a1.Results The expression of BMP9 in nucleus pulposus cells infected with Ad-BMP9 was significantly increased,while the endogenous BMP9 was not expressed in NPCs.The proliferation of NPCs in the experimental group was statistically different from that in the control group,and the G1 phase of the cells was significantly shortened,the S phase was prolonged,and the G2-M phase was also slightly increased.The expression of aggrecan and Col2a1 increased significantly in the experimental group,especially in the increase of aggrecan.Conclusion BMP9 can promote the proliferation of NPCs and the expression of ECM in NPCs.PART3.THE RELATIONSHIP BETWEEN BMP9 AND NOTCH SIGNALING PATHWAY AND THE REGULATION OF NOTCH SIGNALING IN NPCS BY BMP9Objective To investigate the relationship between BMP9 and Notch signaling pathway and to understand the changes of receptors and ligands in Notch signaling after BMP9 intervention in nucleus pulposus cells.Methods RT-PCR and Western blot were used to detect the expression of key molecules and target genes of Notch signaling pathway in nucleus pulposus cells after BMP9 intervention.The changes of Notch receptor and ligand in nucleus pulposus cells after BMP9 intervention were detected by RT-PCR.Results It was shown that the key molecules of Notch signaling pathway,NICD-1(a activation form of NICD)and target genes hes1 and hes5 and hey2,were decreased after BMP9 intervention,and hey1,hey L no significant difference.The expression of Jagged1 was the highest in the ligands of Notch signaling in untreated NPCs.Jagged2 and DLL3 were not found,and each receptor was expressed in different degrees,among which Notch3 was the most expressed.After BMP9 intervention,Notch1 and DLL1 in nucleus pulposus cells decreased significantly,Notch4 was slightly up-regulated,while the other receptors and ligands did not change significantly.Conclusion BMP9 is closely related to Notch signaling.PART4.REGULATION OF ECM BY DNNOTCH1,DLL1 AND JAGGED1 IN NPCSObjective To investigate the effects of dnNotch1,DLL1 and Jagged 1on ECM of NPCsMethod dnNotch1,DLL1,Jagged1 were grouped as follows:(1)Ad-GFP+Ad-RFP,Ad-BMP9+Ad-RFP,Ad-GFP+Ad-dnNotch1,Ad-BMP9+Ad-dnNotch1;(2)Ad-GFP+Ad-RFP,Ad-BMP9+Ad-RFP,Ad-GFP+Ad-DLL1,Ad-BMP9+Ad-DLL1;and(3)Ad-GFP+Ad-RFP,Ad-BMP9+Ad-RFP,Ad-GFP+Ad-Jagged1,Ad-BMP9+Ad-Jagged1.Three groups of viruses infected to NPCs by the above combinations,and the expression levels of aggrecan and Col2a1 were detected by RT-PCR and Western blot,and compared within the group.Results DLL1 group: The m RNA expression of aggrecan and Col2a1:Ad-BMP9+Ad-RFP> Ad-GFP+Ad-RFP>Ad-GFP+Ad-DLL1、Ad-BMP9+Ad-DLL1(P < 0.05).Ad-GFP+Ad-DLL1 and Ad-BMP9+Ad-DLL1 no significant difference(P>0.05);The protein expression of aggrecan and Col2a1:Ad-BMP9+Ad-RFP> Ad-GFP+Ad-RFP、Ad-GFP+Ad-DLL1、Ad-BMP9+Ad-DLL1(P < 0.05),Ad-GFP+Ad-DLL1 and Ad-BMP9+Ad-DLL1 no significant difference.aggrecan :Ad-GFP+Ad-RFP>Ad-GFP+ Ad-DLL1(P<0.05),Ad-GFP+Ad-RFP and Ad-BMP9+Ad-DLL1 no significant difference(P > 0.05).Col2a1 :Ad-GFP+Ad-RFP 、 Ad-GFP+Ad-DLL1 and Ad-BMP9+Ad-DLL1 no significant difference(P>0.05).Jagged1 group: The m RNA expression of aggrecan and Col2a1 :Ad-BMP9+Ad-RFP>Ad-GFP+Ad-RFP>Ad-GFP+Ad-Jagged1 、Ad-BMP9+Ad-Jagged1(P < 0.05).Ad-GFP+Ad-Jagged1 and Ad-BMP9+Ad-Jagged1 no significant difference(P>0.05);The protein expression of aggrecan:Ad-BMP9+Ad-RFP>Ad-GFP+Ad-RFP>Ad-GFP+Ad-Jagged1(P< 0.05).Ad-BMP9+Ad-Jagged1 and Ad-GFP+Ad-RFP 、 Ad-GFP+AdJagged1 and Ad-BMP9+Ad-Jagged1 no significant difference(P>0.05);The protein expression of Col2a1:Ad-BMP9+Ad-RFP>Ad-GFP+AdRFP>Ad-BMP9+Ad-Jagged1>Ad-GFP+Ad-Jagged1(P<0.05).dnNotch1 group: The m RNA expression of aggrecan and Col2a1 :Ad-BMP9+Ad-dnNotch1>Ad-GFP+Ad-dnNotch1>Ad-BMP9+Ad-RFP>A d-GFP+Ad-RFP were statistically different(P < 0.05);The protein expression of aggrecan and Col2a1:Ad-BMP9+Ad-dnNotch1、Ad-GFP+Ad-dnNotch1>Ad-BMP9+Ad-RFP>Ad-GFP+Ad-RFP(P < 0.05).aggrecan : Ad-BMP9+Ad-dnNotch1>Ad-GFP+Ad-dnNotch1 was statistically different(P < 0.05).Col2a1 : Ad-BMP9+Ad-dnNotch1 and Ad-GFP+Ad-dnNotch1 no significant difference(P>0.05).Conclusion dnNotch1 can significantly promote the expression of aggrecan and Col2a1 in NPCs,while DLL1 and Jagged1 have opposite inhibitory effects.