摘要
目的 探讨8BrcAMP对人视网膜母细胞瘤HXORb44细胞抑癌基因表达的效应。方法 应用RNA斑点印迹技术检测细胞p16、p21wafl、wp53、mp53和Rb的mRNA,应用免疫组化斑点印迹技术检测细胞P16、P21wafl、PRb、cdk2、cdk4和PCNA蛋白质表达的免疫反应性(IR)。结果 在斑点印迹标本上,人HXORb44细胞p16、p21wafl、wp53及Rb的mRNA信号和P16IR、P21waflIR及PRbIR均是实验组高于对照组,P<005～001,而mp53mRNA信号、PCNAIR、cdk2IR和cdk4IR均是实验组低于对照组,P<005～001。结论 8BrcAMP上调人HXORb44细胞p16、p21wafl、wp53、Rb抑癌基因的表达及P16、P21wafl、PRb蛋白的表达,并下调mp53mRNA、PCNAIR、cdk2IR和cdk4IR的表达,提示8BrcAMP可通过阻抑细胞周期进展相关基因的表达而抑制人HXORb44细胞的生长增殖。
ObjectiveIt is known that 8 Br cAMP is one of selective binding site analogues for cAMP RIIα to affect cell growth through regulation of gene expression.The p16,p21wafl,p53 and Rb are antioncogenes which affect cell growth through control of cell cycle.The aim of this study is to investigate the 8 Br cAMP effect on the expression of antioncogenes in human HXO Rb44 cells.Methods Cultured HXO Rb44 cells in RPMI 1640 medium were divided into two aliquots.8 Br cAMP (2×10 -5 mol/L) was added into one aliquot for 24?h as the experimental group(EG),the another aliquot without 8 Br cAMP as the control group(CG).After 24?h,the cell suspension was dropped onto the nitrocellulose membrane.The mRNA of p16,p21wafl,wild type(w)p53,mutant type(m) p53 and Rb were used respectively with biotin labeled cDNA probes by intact cell RNA dot blot.The immunoreactivity(IR) of P16,P21wafl,PRb,PCNA,cdk2 and cdk4 were detected respectively with specific monoclonal antibodies on dot blot.ResultsThe mRNA dot blot signals of mp53 and protein dot blot of cdk2 IR,cdk4 IR and PCNA IR in EG were weaker than those in CG( P <0 05～0 01).While,the mRNA signals of p16,p21wafl,wp53 and Rb in EG were stronger than those in CG( P <0 05～0 01).The intensity of each protein dot blot was consistent with that of their RNA dot blot (except for wP53 IR and mP53 IR not to be done).Conclusions (1)8 Br cAMP could up regulate expression of antioncogenes including p16,p21wafl,wp53,Rb,and protein expression of P16,P21wafl and PRb.(2)8 Br cAMP could down regulate mp53 gene expression and protein expression of cdk2,cdk4 and PCNA.The results suggest that 8 Br cAMP could inhibit human HXO Rb44 cell growth through interfering related gene expression of cell cycle.
出处
《眼科研究》
CSCD
2000年第1期1-3,共3页
Chinese Ophthalmic Research
