Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C（IL-17RC）,a phenomenon observed in peripheral blood and chorioretinal tissues with age-rela...Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C（IL-17RC）,a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration（AMD）,was associated with altered activation of phosphatidylinositide 3-kinase（PI3K）,Akt,and glycogen synthase kinase 3（GSK3）.We wondered whether or not altered PI3 K,Akt,and GSK3 activities could be detected in peripheral blood mononuclear cells（PBMC） obtained from AMD patients.In the patients＇ PBMC,absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed,which was accompanied with increased phosphorylation of GSK3 substrates（e.g.CCAAT enhancer binding protein a,insulin receptor substrate 1,and TAU）,indicative of enhanced GSK3 activation.In addition,decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients,suggesting impaired PI3 K activation.Moreover,abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients.These data demonstrate that despite the presence of high levels of IL-17 RC,Wnt-3a and vascular endothelial growth factor,the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients.Thus,measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.展开更多
Phosphatidylinositide 3-kinase （PI3K）/protein kinase B （PKB, Akt） pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely ap- plied as an inhibito...Phosphatidylinositide 3-kinase （PI3K）/protein kinase B （PKB, Akt） pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely ap- plied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 ceils was investigated. The expression of PI3K, phosphorylated AM （p-Akt） and multidrng-resistance like protein （MRP） in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was de- tected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and pro- tein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pan- creas tissues and chronic pancreatitis tissues （P〈0.01 and P〈0.05 respectively）. LY294002 could effec- tively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.展开更多
[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋...[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋白表达,CCK-8、流式细胞术检测细胞增殖、凋亡情况,Western Blotting检测磷脂酸肌醇-3-激酶(PI3K)、丝苏氨酸蛋白激酶(AKT)通路蛋白表达。[结果]sh/Cx43-1组、sh/Cx43-2组Cx43 mRNA或蛋白均低于sh/Con组,且sh/Cx43-1组低于sh/Cx43-2组(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05);培养24h时各组细胞增殖水平无明显差异(P>0.05),培养48 h、72 h时sh/Cx43-1组、sh/Cx43-2组细胞增殖水平均低于sh/Con组(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);sh/Cx43-1组、sh/Cx43-2组细胞总凋亡率高于sh/Con组,PI3K、AKT蛋白表达均低于sh/Con组,且sh/Cx43-1组变化较sh/Cx43-2组更明显(P<0.05)。[结论]通过shRNA靶向敲低Cx43基因表达能显著抑制MM.1S细胞增殖,促进其凋亡,这可能与敲低Cx43后PI3K/AKT通路受到抑制有关。展开更多
基金supported by intramural research funding of National Center for Complementary and Alternative Medicine(now is National Center for Complementary and Integrative Health),NIH,the US Department of Health and Human Services(to X.L.)and an operating grant(MOP 123279)from Canadian Institutes for Health Research(to Z.Y.)
文摘Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C（IL-17RC）,a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration（AMD）,was associated with altered activation of phosphatidylinositide 3-kinase（PI3K）,Akt,and glycogen synthase kinase 3（GSK3）.We wondered whether or not altered PI3 K,Akt,and GSK3 activities could be detected in peripheral blood mononuclear cells（PBMC） obtained from AMD patients.In the patients＇ PBMC,absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed,which was accompanied with increased phosphorylation of GSK3 substrates（e.g.CCAAT enhancer binding protein a,insulin receptor substrate 1,and TAU）,indicative of enhanced GSK3 activation.In addition,decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients,suggesting impaired PI3 K activation.Moreover,abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients.These data demonstrate that despite the presence of high levels of IL-17 RC,Wnt-3a and vascular endothelial growth factor,the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients.Thus,measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.
文摘Phosphatidylinositide 3-kinase （PI3K）/protein kinase B （PKB, Akt） pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely ap- plied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 ceils was investigated. The expression of PI3K, phosphorylated AM （p-Akt） and multidrng-resistance like protein （MRP） in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was de- tected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and pro- tein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pan- creas tissues and chronic pancreatitis tissues （P〈0.01 and P〈0.05 respectively）. LY294002 could effec- tively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.
文摘[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋白表达,CCK-8、流式细胞术检测细胞增殖、凋亡情况,Western Blotting检测磷脂酸肌醇-3-激酶(PI3K)、丝苏氨酸蛋白激酶(AKT)通路蛋白表达。[结果]sh/Cx43-1组、sh/Cx43-2组Cx43 mRNA或蛋白均低于sh/Con组,且sh/Cx43-1组低于sh/Cx43-2组(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05);培养24h时各组细胞增殖水平无明显差异(P>0.05),培养48 h、72 h时sh/Cx43-1组、sh/Cx43-2组细胞增殖水平均低于sh/Con组(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);sh/Cx43-1组、sh/Cx43-2组细胞总凋亡率高于sh/Con组,PI3K、AKT蛋白表达均低于sh/Con组,且sh/Cx43-1组变化较sh/Cx43-2组更明显(P<0.05)。[结论]通过shRNA靶向敲低Cx43基因表达能显著抑制MM.1S细胞增殖,促进其凋亡,这可能与敲低Cx43后PI3K/AKT通路受到抑制有关。
