Inhibition Of Insulin-like Growth Factor-â…  On Apoptosis And Effects Of The Intracellar Calcium Ion In Articular Chondrocytes Of Rabbit In Vitro

1,Isolation and culture of the chondrocytes from articular cartilage of the rabbit and study on the biological behaviors in vitroObjective: To culture the articular chondrocytes of the rabbits in vitro. Methods: The articular cartilage from neonatel rabbit was dissecteded ,cut into the size of 1mm³ or so and then digested in 0.2% collagenase â…¡ solution for 5 hours. The isolated cells were cultured in DMEM medium and identified with type â…¡ collagen immunohistochemistry reaction and Giemsa staining.Result: When stained with toluidine blue and anti-type â…¡ collagen antibody, the results were positive. Conclusion: The methods for isolation and culture of chondrocytes is simple and feasible. The growth of passage one and passage two cells are quite well and suitable for experiments. 2,Establishment of Two Models on Chondrocyte ApoptosisObjective: To investingate the effect of drug on apoptosis of chondrocyte in osteoarthritis, the twochondrocyte apoptosis models were established. Methods: treated with sodium nitroprusside( SNAP) and all-rans retinoic acid(ATRA) respectively. The apoptosis rate were analysed by Annexin V/PT with flow cytometry and the characteristic alterations of morphology were detected by AO staining,DNA ladder and electron miscroscope. Result: When rabbit chondrocyte were incubated with SNAP(2mmol/L) and ATRA(10^-4 mmol/L) for 24h, they all could induce apoptosis in chondrocyte in vitro. Conclusion: The results suggested that the chondro cyte apoptosis models could be established by sodium nitroprusside and all-trans retinoic acid. 3,Inhibition of insulin-like growth factor-I on apoptosis of articular chondrocytesObjective: To study the effects of insulin-like growth factor- 1(IGF-I) and apoptosis of rabbit articular chondrocytes induced by SNAP,ATRA and the expression of bcl-2and caspase-3. Methods: Two Models on Chondrocyte Apoptosis was set up. IGF-I 10ug/L,100ug/L added into the culture, and the apoptosis of chondrocyte was assessed by TUNEL staining and bcl-2 and caspase-3 immunohistochemical staining. Result: Compared with apoptosis group, the number of apoptotic cells and the expression of caspase-3 positive cells in IGF-I treated group were decreased significantly. The expression of bcl-2 positive cells was increased significantly. Conclusion: IGF-I can decrease apoptosis by up-regulatinn bcl-2 and down-regulating caspase-3.4, Antagonistic effects of insulin-like growth factor-1 (IGF-I) on [Ca²⁺]I mobilization by SNAP,ATRA in newborn rabbit chondrocyteAbstract Object: To study the effects of insulin-like growth factor-1 (IGF-I) on intracellular calcium concentration([Ca²⁺]i) mobilized by SNAP,ATRA. Methods: [Ca²⁺]I was measured with fluorescent intensity (FI) by using fluorescent indicator,Fura-2/AM and calculated with the formula: [Ca²⁺]I=keff×(R-Rmin)/(Rmax-R). Results: 100ug/L IGF-I significant inhibited the Ca²⁺influx induced by ATRA 10^-4mol/Land SNAP . 2mmol/L(P<0.01). Conclusions: ATRA and SNAP can induce apoptosis in chondricytes in vitro, but IGF-I possessed the antagonistic effecys on [Ca²⁺]I increases in newborn rabbit chondrocyte.