The Expression And Biological Effects Of CCN3 In Cellular Apoptosis Model Induced By Serum Deprivation In Rat Endplate Chondrocytes In Vitro

PART ONE Establishment of an apoptotic model of cartilage endplate cells and detection of CCN3 expression in vitroObjective:To determine the apoptotic effects of cartilage endplate (CEP) cells induced by serum deprivation and detect the CCN3 expression in the apoptotic model in vitro.Methods:Rat CEP cells were isolated, cultured in monolayers and confirmed by toluidine blue staining. After incubated in either 1% or 10% fetal bovine serum (FBS) for 48 h. DAPI and Annexin V/PI staining to utilized to qualify and quantify the cells apoptosis. Apoptosis associated protein Fas, cytochrome C, caspase3 and PARP were detected by Western Blot, β-actin was used as a control to verify equal protein loading Activated caspases (-3,-8, and-9) were detected to determine the apoptotic pathway. CCN3 mRNA expression was detected by RT-PCR.Results:Cells cultured from the CEP were viable and maintain a chondrocytic phenotype confirmed by toluidine blue staining. DAPI positive cells increased significantly, the apoptosis rate quantified by FCM was greatly increased in 1% FBS compared with 10% FBS (10.06%±0.35% VS 22.3%±0.58%, p<0.05). Western Blot results showed that the expression of Fas protein, cytochrome C, caspase3 and PARP was significantly up-regulated. Caspase-3,-8,-9 enzyme activities were increased by 4 times,3 times and 4 times, respectively. CCN3 mRNA expression were also significantly increased by 5 folds compared with that in 10% FBS.Conclusion:The apoptosis of CEP cells could be induced by serum deprivation, and CCN3 mRNA expression were upregulated in vitro. PART TWOThe biological effect of CCN3 on apoptosis and matrix secretion of CEP cells in serum deprivation in vitro.Objective:To explore the biologic effects of CCN3 on CEP cells apoptosis and matrix secretion in vitro.Methods:Rat CEP cells were isolated, cultured in monolayers; two groups cells cultured with concentration gradient rCCN3 (Ong/mL, 100ng/mL,200ng/mL, 400ng/mL) in 10%FBS or 1%FBS, cells were stained by Annexin V/PI and the apoptosis rate was quantified by FCM after 48h. Cells were cultured with 100ng/mL rCCN3 or 100ng/mL rCCN2 in 10%FBS, the aggrecan and collagen2 mRNA expression were detected at 24h and 48h, so did the another CCN protein mRNA expression.Results:FCM analysis showed that:no obvious differences of apoptosis rate were investigated with the concentration gradient of rCCN3 in 10% FBS group, and the results have no statistically significant (P> 0.05); while in 1% FBS cultured cells, with the increase of rCCN3 concentration, the apoptosis rate was significantly increased (P<0.05). The mRNA expression of aggrecan, collagen2 and CCN2 were significantly inhibited in CCN3 group; however, rCCN2 treatment cells showed an increase aggrecan and collagen2 mRNA expression, but the expression of CCN3 mRNA was significantly inhibited.Conclusion:CCN3 can induce the CEP cells apoptosis in low serum conditions; CCN3 can decrease the secretion of extracellular matrix, whereas CCN2 appears to act antagonistically to CCN3.