The effects of CD44 siRNAon sodium nitroprusside -induced chondrocyte apoptosis protected by carboxymethyl -chitosan
10.3760/cma.j.issn.1007-7480.2011.10.010
- VernacularTitle:小干扰RNA特异性沉默CD44基因对羧甲基壳聚糖保护大鼠软骨细胞凋亡的影响
- Author:
Qing CHEN
;
Bin HE
;
Shiqing LIU
- Publication Type:Journal Article
- Keywords:
Chondrocyte;
Apoptosis;
Antigens,CD44;
RNA,small interfering;
Carboxymethyl chitosan
- From:
Chinese Journal of Rheumatology
2011;15(10):698-702
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the effects of carboxymethyl-chitosan(CMCS) on chondrocyte apoptosis induced by sodium nitroprusside (SNP),and the effects of CD44 in the process.MethodsA small interference RNA (siRNA) targeting to CD44 mRNA (siRNA-1,siRNA-2,siRNA-3) was constructed.The siRNA was transfected into chondrocytes in vitro with LipofectamineTM 2000.The efficacy of transfection was detected by transfecting fluorescence siRNA into cells.The mRNA expression of CD44 in vitro was detected by RT-PCR.The protein level of CD44 was detected by Western blotting.The apoptosis rate of the transfected and non-transfected cells induced by SNP was detected by flow cytometry.Statistical analysis was conducted with one-way ANOVA and SNK-q test.ResultsThe efficacy of transfection was about 60％.As compared with the control group,the mRNA expression was specifically inhibited after transfecting CD44 siRNA-1 for 24,48 and 72 h(0.198±0.007 vs 0.429±0.053 at 24 h,0.211±0.016 vs 0.501±0.037 at 48 h,0.153±0.005 vs 0.341±0.009 at 72h,q=5.93,7.01,11.23,P＜0.01 ),and the protein level of cells was inhibited after transfecting CD44 siRNA-1 for 24 h compared with the control group (0.231±0.064 vs 0.675±0.113,q=13.09,P＜0.01 ).The FCM results showed that 3 mmol/L SNP could induce chondrocytes apoptosis(70±6)％,and 50,100,200 μg/ml C MCS could affect the inhibitory effect of SNP-induced apoptosis of chondrocyte [ (51 ±7)％,(30±4)％,(15±4)％,q=5.08,6.97,9.73,P＜0.01 ],but it had milder inhibitory effect on CD44-siRNA-1 transfected chondrocytes when compared with those of the non-transfected chondrocytes [ (34±6)％ vs(15±4)％,q=6.95,P＜0.01 ].ConclusionThe data of this study has suggest that siRNA-1 against CD44 gene can significantly inhibit the expression of CD44 in chondrocyte of rats in vitro after transfection.The CD44 may play an important role in chondrocyte apoptosis induced by SNP and protected by CMCS.
