Regulation Of Degenerative Intervertebral Disc Nucleus Pulposus Cells Apoptosis By Sirt1and Its Signal Transduction Mechanism

Objective To master the technique of primary and passage culture ofhuman degenerative, normal intervertebral disc (IVD) nucleus pulposus(NP) cells.Methods In short, NP samples were separated from annulus,minced after harvest under sterile conditions. matrix was digested20minutes at37℃in0.25%trypsin solution, following,3-4hours in0.2%type II collagenase. ntaining15%fetal bovine serum (FBS). Monolayercell cultures were maintained in a5%CO2:95%air incubator at37℃.Then, the verification of human NP cells was performed by immunocytochemistry detection using COL2A1antibody. Lastly, MTTassay was used to analyse proliferative activity of NP clls.Results Primary and passage culture of human IVD NP cells derivedfrom patients (>55years old) with lumbar disc herniation (LDH), patients(<25years old) with lumbar vertebra fracture (LVF) were successfullyperformed. Human NP cells were verified according to its characterizationunder microscope and immunocytochemistry staining using COL2A1antibody. Proliferative activity of NP cells of passage2from LVF on thesecond, the third, the fourth day was significantly higher than that of NPcells from LDH (p<0.05).Conclusion we are familiar with the technique of pimary, passageculture of degenerative and normal human IVD NP cells, laying firmfoundation for following investigation. Objective To clarify the expression difference of SIRT1betweendegenerative NP cells and normal NP cells. Methods The gene and protein expression of SIRT1, COL2A1,aggrecan in degenerative, normal NP tissues and cells were investigate byusing immunohistochemical analysis, reverse transcription PCR (RT-PCR),real time RT-PCR, western blot. Apoptotic cells in the human NP surgicalsamples were assessed with a TUNEL assay kit (Roche) that enzymaticallylabels DNA strand breaks.Results Immunohistochemical detection using specific antibodiesrevealed that SIRT1postive cells in degenerative NP sample weresignificantly decreased compared with those in the NP samples frompatient with LVF (p<0.05). Analysis of SIRT1/COL2A1/aggrecan mRNAand protein in human NP cells by PCR and Western blot was alsoperformed to confirm elevated SIRT1levels in NP cells from patients withLVF (p<0.05). The incidence of apoptotic cell of surgical NP specimensfrom patients with LDH was significantly greater compared with thecontrol (p<0.05).Conclusion Expression levels of SIRT1in human NP graduallydecrease with increasing age and degeneration. there is an inverserelationship between SIRT1and cellular apoptosis in human IVD NP cells. Objective To investigate whether SIRT1could regulate apoptosis ofdegenerative NP cells and its molecular mechanism.Methods Apoptotic degenerative NP cells followed SIRT1plasmidtransfection, SIRT1activator-resveratrol, SIRT1-siRNA transfection,SIRT1inhibitor-nicotinamide (NAM) treatment with or without TNFα,low serum conditions (0.5%FBS) were detected by Annexin V/PI doublestaining flow cytometry. We also tried to explore the signaling moleculesthat mediate regulation property of SIRT1by Western blot and PI3Kinhibitor-LY294002analysis.Results SIRT1plasmid transfection experiments by lipofection2000or PolyJet did not be successfully conducted because of extremely lowtransfection efficiency. The rate of apoptosis was far fewer inresveratrol-treated NP cells with or without TNFα, low serum conditions(0.5%FBS) than control (p<0.05). However, SIRT1siRNA transfected ornicotinamide-treated NP cells significantly increased degenerative humanNP cells apoptosis rate detected by Annexin V/PI double staining flow cytometry than control (p<0.05). After SIRT1siRNA transfected, NP cellsdecreased phosphorylation of Akt, while resveratrol phosphorylated Akt.Treatment with LY294002or Akt siRNA increased the rate of apoptosis.Conclusion SIRT1plays a critical role in survival of degenerativehuman NP cells through the Akt anti-apoptotic signaling pathway.