AIM:To investigate the effects of small interfering RNA(siRNA)-mediated inhibition of Class Ⅰ phosphoinositide 3-kinase(Class Ⅰ PI3K) signal transduction on the proliferation,apoptosis,and autophagy of gastric cance...AIM:To investigate the effects of small interfering RNA(siRNA)-mediated inhibition of Class Ⅰ phosphoinositide 3-kinase(Class Ⅰ PI3K) signal transduction on the proliferation,apoptosis,and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS:We constructed the recombinant replication adenovirus PI3K(I)-RNA interference(RNAi)-green fluorescent protein(GFP) and control adenovirus NCRNAi-GFP,and infected it into human gastric cancer cells.MTT assay was used to determine the growth rate of the gastric cancer cells.Activation of autophagy was monitored with monodansylcadaverine(MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment.Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3).Mitochondrial membrane potential was measured using the fluorescent probe JC-1.The expression of autophagy was monitored with MDC,LC3 staining,and transmission electron microscopy.Western blotting was used to detect p53,Beclin-1,Bcl-2,and LC3 protein expression in the culture supernatant.RESULTS:The viability of gastric cancer cells was inhibited after siRNA targeting to the Class Ⅰ PI3K blocked Class Ⅰ PI3K signal pathway.MTT assays revealed that,after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP,the rate of inhibition reached 27.48% ± 2.71% at 24 h,41.92% ± 2.02% at 48 h,and 50.85% ± 0.91% at 72 h.After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAiGFP,the rate of inhibition reached 24.39% ± 0.93% at 24 h,47.00% ± 0.87% at 48 h,and 70.30% ± 0.86% at 72 h(P < 0.05 compared to control group).It was determined that when 50 MOI,the transfection efficiency was 95% ± 2.4%.Adenovirus PI3K(I)RNAi-GFP(50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells,and the results described here prove that RNAi of Class Ⅰ PI3K induced apoptosis in SGC7901 cells.The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity,indicating increased formation of autophagosomes.The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low.After incubating with adenovirus PI3K(I)-RNAi-GFP(50 MOI),Beclin-1,LC3,and p53 protein expression was significantly increased from 24 to 72 h.We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP(50 MOI).A number of isolated membranes,possibly derived from ribosomefree endoplasmic reticulum,were seen.These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles.We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)RNAi-GFP(50 MOI) treatment.Control cells showed a round shape and contained normal-looking organelles,nucleus,and chromatin,while adenovirus PI3K(I)-RNAiGFP(50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION:After the Class Ⅰ PI3K signaling pathway has been blocked by siRNA,the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.展开更多
AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA excl...AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.展开更多
基金Supported by The Natural Science Foundation of China,No. 81172348Suzhou High-Level Talents Project,2008-11+1 种基金Suzhou Science and Technology Development Foundation,2010SYS201031the Science,Education,and Health Foundation of Suzhou City,SWKQ0914 and SWKQ0916
文摘AIM:To investigate the effects of small interfering RNA(siRNA)-mediated inhibition of Class Ⅰ phosphoinositide 3-kinase(Class Ⅰ PI3K) signal transduction on the proliferation,apoptosis,and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS:We constructed the recombinant replication adenovirus PI3K(I)-RNA interference(RNAi)-green fluorescent protein(GFP) and control adenovirus NCRNAi-GFP,and infected it into human gastric cancer cells.MTT assay was used to determine the growth rate of the gastric cancer cells.Activation of autophagy was monitored with monodansylcadaverine(MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment.Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3).Mitochondrial membrane potential was measured using the fluorescent probe JC-1.The expression of autophagy was monitored with MDC,LC3 staining,and transmission electron microscopy.Western blotting was used to detect p53,Beclin-1,Bcl-2,and LC3 protein expression in the culture supernatant.RESULTS:The viability of gastric cancer cells was inhibited after siRNA targeting to the Class Ⅰ PI3K blocked Class Ⅰ PI3K signal pathway.MTT assays revealed that,after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP,the rate of inhibition reached 27.48% ± 2.71% at 24 h,41.92% ± 2.02% at 48 h,and 50.85% ± 0.91% at 72 h.After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAiGFP,the rate of inhibition reached 24.39% ± 0.93% at 24 h,47.00% ± 0.87% at 48 h,and 70.30% ± 0.86% at 72 h(P < 0.05 compared to control group).It was determined that when 50 MOI,the transfection efficiency was 95% ± 2.4%.Adenovirus PI3K(I)RNAi-GFP(50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells,and the results described here prove that RNAi of Class Ⅰ PI3K induced apoptosis in SGC7901 cells.The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity,indicating increased formation of autophagosomes.The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low.After incubating with adenovirus PI3K(I)-RNAi-GFP(50 MOI),Beclin-1,LC3,and p53 protein expression was significantly increased from 24 to 72 h.We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP(50 MOI).A number of isolated membranes,possibly derived from ribosomefree endoplasmic reticulum,were seen.These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles.We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)RNAi-GFP(50 MOI) treatment.Control cells showed a round shape and contained normal-looking organelles,nucleus,and chromatin,while adenovirus PI3K(I)-RNAiGFP(50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION:After the Class Ⅰ PI3K signaling pathway has been blocked by siRNA,the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.
文摘AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.
