CAJ | 학술논문

目的通过观察肼苯哒嗪、5'-氮杂-2'-脱氧胞嘧啶核苷酸(5'-aza-2'-deoxycytidine,5-Aza-CdR)及其联合使用对人成骨肉瘤细胞MG-63的生长抑制作用,并检测对骨肉瘤细胞中WWOX基因表达的调控作用,初步明确肼苯哒嗪对骨肉瘤细胞的作用机制.方法取对数生长期的人骨肉瘤细胞株MG-63制成细胞悬液,计数板计数5×104个/ml,加入96孔板.分为肼苯哒嗪组(药物浓度为0.1、1.0、10 μmol/L)、5-Aza-CdR组(药物剂量为5、10、20 μmol/L)、肼苯哒嗪联合5-Aza-CdR组(药物剂量为0.1 μmo/L+5 μmo/L、1.0 lmol/L+10 μlmol/L、10 μmol/L+20 μmol/L)及对照组(培养基).噻唑蓝(MTT)比色法检测药物对MG-63细胞生长抑制作用;流式细胞仪检测药物对细胞周期分布作用及凋亡影响;Real-Time PCR技术检测WWOX mRNA表达改变情况;Western-blot检测药物处理后各组细胞中WWOX蛋白表达水平.结果肼苯哒嗪、5-Aza-CdR及其联合应用对MG-63细胞的增殖均有明显抑制作用,且呈浓度依赖性并与作用时间相关;联合应用较单用抑制作用明显增强,且各组差异有统计学意义.肼苯哒嗪、5-Aza-CdR可诱导骨肉瘤MG-63细胞凋亡,联合应用时诱导凋亡作用加强.肼苯哒嗪、5-Aza-CdR对骨肉瘤MG-63细胞中WWOX基因表达有促进作用,联合作用使WWOX表达明显增强.结论肼苯哒嗪可明显抑制骨肉瘤细胞的增殖并诱导其凋亡,并促进WWOX基因的表达,其作用机制可能为肼苯哒嗪能够使抑癌基因WWOX基因甲基化状态逆转,表达增加,从而抑制MG-63细胞的增殖.
목적 통과관찰정분달진、5'-담잡-2'-탈양포밀정핵감산(5'-aza-2'-deoxycytidine,5-Aza-CdR)급기연합사용대인성골육류세포MG-63적생장억제작용,병검측대골육류세포중WWOX기인표체적조공작용,초보명학정분달진대골육류세포적작용궤제.방법 취대수생장기적인골육류세포주MG-63제성세포현액,계수판계수5×104개/ml,가입96공판.분위정분달진조(약물농도위0.1、1.0、10 μmol/L)、5-Aza-CdR조(약물제량위5、10、20 μmol/L)、정분달진연합5-Aza-CdR조(약물제량위0.1 μmo/L+5 μmo/L、1.0 lmol/L+10 μlmol/L、10 μmol/L+20 μmol/L)급대조조(배양기).새서람(MTT)비색법검측약물대MG-63세포생장억제작용;류식세포의검측약물대세포주기분포작용급조망영향;Real-Time PCR기술검측WWOX mRNA표체개변정황;Western-blot검측약물처리후각조세포중WWOX단백표체수평.결과 정분달진、5-Aza-CdR급기연합응용대MG-63세포적증식균유명현억제작용,차정농도의뢰성병여작용시간상관;연합응용교단용억제작용명현증강,차각조차이유통계학의의.정분달진、5-Aza-CdR가유도골육류MG-63세포조망,연합응용시유도조망작용가강.정분달진、5-Aza-CdR대골육류MG-63세포중WWOX기인표체유촉진작용,연합작용사WWOX표체명현증강.결론 정분달진가명현억제골육류세포적증식병유도기조망,병촉진WWOX기인적표체,기작용궤제가능위정분달진능구사억암기인WWOX기인갑기화상태역전,표체증가,종이억제MG-63세포적증식.
Objective To investigate the growth inhibition of human osteosareoma cell line(MG-63) intervened by Hydralazine and 5'-aza-2'-deoxycytidine (5-Aza-CdR),and the effect on the mRNA expression of gene WW domain-containing oxidoreductase (WWOX).Methods Certain volume of 5 × 104/ml of human osteosarcoma cell line MG-63 in logarithmic growth phase were added into 96-well plate.There were Hydralazine group (drug concentration,0.1,1.0,10 μmol/L),5-Aza-CdR group (drug concentration,5,10,20 μmol/L),Hydralazine combined with 5-Aza-CdR group (drug concentration,0.1 μmo/L + 5 μmol/L,1.0 μmol/L + 10 μmol/L,10 μmol/L + 20 μmol/L) and control group (culture medium).Methyl thiazol tetrazolium(MTT) colorimetric methods were used to test the growth inhibition of MG-63 cells intervened by different concentrations of Hydralazine and 5'-aza -2'-deoxycytidine (5-Aza-CdR).Flow cytometry AnnexinV-FITC/PI methods were used to assay the effects of Hydralazine and 5-Aza-CdR inducing apoptosis in osteosarcoma cells in vitro.Real-time polymerase chain reaction (Real-Time PCR)methods were used to detect amplification of WWOX mRNA induced by Hydralazine combined with 5-Aza-CdR or alone.Western-blotting methods were used to examine the expression of WWOX in MG-63 cells.Results Hydralazine and 5-Aza-CdR effectively inhibited the growth of MG-63 cells in a concentration and time-dependent manner.Combined effect was more obvious.Further more the expression levels of WWOX mRNA and protein were increased significantly in combined groups as compared with other groups.Conclusion Hydralazine and 5-Aza-CdR could effectively inhibit the proliferation of MG-63 cells and induce apoptosis which is concurrent with the promotion of the expression of WWOX.The mechanism may be that Hydralazine/5-Aza-CdR effectively cause the demethylated of WWOX gene CpG-rich promoter regions,leading to the high expression of WWOX and inhibit the growth of MG-63 cells.The use of hydralazine in the treatment of osteosarcoma is worthy of further investigation.