[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
In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the ...In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins(1,4,5)P3 concentration at the apical region of pollen tube, the caged Ins(1,4,5)P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain (10 μm from the tip). More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins(1,4,5)P3 concentration (n=21). While, pollen tubes without being loaded with caged Ins(1,4,5)P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins(1,4,5)P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins(1,4,5)P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins(1,4,5)P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins(1,4,5)P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins(1,4,5)P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.展开更多
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。
文摘In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins(1,4,5)P3 concentration at the apical region of pollen tube, the caged Ins(1,4,5)P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain (10 μm from the tip). More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins(1,4,5)P3 concentration (n=21). While, pollen tubes without being loaded with caged Ins(1,4,5)P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins(1,4,5)P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins(1,4,5)P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins(1,4,5)P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins(1,4,5)P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins(1,4,5)P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.