目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并...目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并扩增培养,用逆转录聚合酶链反应法(RT-PCR)检测PLCεmRNA的表达情况,应用Transwell小室侵袭试验、明胶酶谱分析分别研究转染前、后膀胱癌细胞侵袭转移能力的变化,并以空载质粒HK-A转染和未转染细胞为对照。结果:RT-PCR检测显示pll-PLCε,p12-PLCε转染成功后T24细胞PLCεmRNA表达明显比空质粒HK-A转染和未转染细胞减弱;p12-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(26.8±5.8)和pll-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(25.8±6.2)明显少于未转染细胞(34.8±6.9)和HK-A转染细胞(33.8±5.7)(P<0.01);明胶酶谱分析显示转染pll-PLCε、p12-PLCε均使细胞分泌MMP-2、MMP-9明显低于未转染细胞和HK-A转染细胞。结论:推测转染外源性PLCεsiRNA基因能下调PLCε基因的表达并能抑制膀胱癌细胞侵袭转移。展开更多
目的研究磷脂酶Cε(phospholipase C epsilon,PLCε)对人骨肉瘤细胞株U2OS迁移侵袭能力的影响。方法 siRNA干扰U2OS细胞PLCε的表达,应用CCK-8实验检测PLCε对细胞增殖能力的影响,以划痕实验、Transwell小室模型实验测定细胞迁移能力改...目的研究磷脂酶Cε(phospholipase C epsilon,PLCε)对人骨肉瘤细胞株U2OS迁移侵袭能力的影响。方法 siRNA干扰U2OS细胞PLCε的表达,应用CCK-8实验检测PLCε对细胞增殖能力的影响,以划痕实验、Transwell小室模型实验测定细胞迁移能力改变情况,以明胶酶谱实验测定细胞分泌基质金属蛋白酶MMP2的变化。结果体外合成特异性PLCε基因siRNA可明显抑制U2OS细胞中PLCε的表达;沉默PLCε后,U2OS细胞的增殖能力无明显变化,而划痕愈合能力、迁移活力较对照组细胞明显下降,其分泌MMP2的水平也明显下降。结论沉默PLCε可致人骨肉瘤细胞U2OS的迁移和侵袭能力下降。展开更多
The present study aims to explore the effects of p53 and its target gene Rap2B on the autophagy of U2OS cells.U2OS cells were treated with siRNA against p53,Rap2B,and PLCε.Relative expressions of p53,Rap2B,and PLCεw...The present study aims to explore the effects of p53 and its target gene Rap2B on the autophagy of U2OS cells.U2OS cells were treated with siRNA against p53,Rap2B,and PLCε.Relative expressions of p53,Rap2B,and PLCεwere determined using quantitative polymerase chain reaction(qPCR)and Western blotting,respectively.Levels of IP3 in the cells were determined using Enzyme-linked Immunosorbent Assay(ELISA).Levels of Ca^(2+) were detected using Flow cytometry.Fluorescence microscopy was used to observe the autophagy of cells.Knockdown of p53 significantly decreased the expressions of Rap2B protein.Additionally,knockdown of p53 significantly decreased the mRNA levels of PLCε.The knockdown of p53,Rap2B,and PLCεsignificantly decreased the levels of intracellular IP3 and Ca^(2+) and promoted autophagy of U2OS cells.Our results demonstrated that p53-Rap2B-PLCε-IP3 signaling pathway regulated autophagy of U2OS cells.展开更多
磷脂酶Cε1(phospholipase C epsilon-1,PLCE1)基因定位于常染色体10q23.32-q24.1,其编码蛋白为磷脂酶Cε1(phospholipase C epsilon-1,PLCε1)。PLCε1蛋白是最新发现的一种磷脂酶C(phospholipase C,PLC)同工酶,在成熟的肾小球足细胞表...磷脂酶Cε1(phospholipase C epsilon-1,PLCE1)基因定位于常染色体10q23.32-q24.1,其编码蛋白为磷脂酶Cε1(phospholipase C epsilon-1,PLCε1)。PLCε1蛋白是最新发现的一种磷脂酶C(phospholipase C,PLC)同工酶,在成熟的肾小球足细胞表达,参与肾小球毛细血管袢的形成和正常发育。近来发现PLCE1基因突变可以引起常染色体隐性遗传性肾病综合征。PLCE1基因移码或无义突变可导致婴儿早发性肾病综合征,其病理特征为弥漫性肾小球硬化(diffuse mesangial sclerosis,DMS);而错义突变可导致局灶性节段性肾小球硬化(focal segmental glo-merulosclerosis,FSGS)。但其确切发病情况及机制目前尚不清楚,本文就PLCE1基因及其编码蛋白与激素耐药性肾病综合征之间的关系作一简要综述。展开更多
文摘目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并扩增培养,用逆转录聚合酶链反应法(RT-PCR)检测PLCεmRNA的表达情况,应用Transwell小室侵袭试验、明胶酶谱分析分别研究转染前、后膀胱癌细胞侵袭转移能力的变化,并以空载质粒HK-A转染和未转染细胞为对照。结果:RT-PCR检测显示pll-PLCε,p12-PLCε转染成功后T24细胞PLCεmRNA表达明显比空质粒HK-A转染和未转染细胞减弱;p12-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(26.8±5.8)和pll-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(25.8±6.2)明显少于未转染细胞(34.8±6.9)和HK-A转染细胞(33.8±5.7)(P<0.01);明胶酶谱分析显示转染pll-PLCε、p12-PLCε均使细胞分泌MMP-2、MMP-9明显低于未转染细胞和HK-A转染细胞。结论:推测转染外源性PLCεsiRNA基因能下调PLCε基因的表达并能抑制膀胱癌细胞侵袭转移。
文摘目的研究磷脂酶Cε(phospholipase C epsilon,PLCε)对人骨肉瘤细胞株U2OS迁移侵袭能力的影响。方法 siRNA干扰U2OS细胞PLCε的表达,应用CCK-8实验检测PLCε对细胞增殖能力的影响,以划痕实验、Transwell小室模型实验测定细胞迁移能力改变情况,以明胶酶谱实验测定细胞分泌基质金属蛋白酶MMP2的变化。结果体外合成特异性PLCε基因siRNA可明显抑制U2OS细胞中PLCε的表达;沉默PLCε后,U2OS细胞的增殖能力无明显变化,而划痕愈合能力、迁移活力较对照组细胞明显下降,其分泌MMP2的水平也明显下降。结论沉默PLCε可致人骨肉瘤细胞U2OS的迁移和侵袭能力下降。
文摘The present study aims to explore the effects of p53 and its target gene Rap2B on the autophagy of U2OS cells.U2OS cells were treated with siRNA against p53,Rap2B,and PLCε.Relative expressions of p53,Rap2B,and PLCεwere determined using quantitative polymerase chain reaction(qPCR)and Western blotting,respectively.Levels of IP3 in the cells were determined using Enzyme-linked Immunosorbent Assay(ELISA).Levels of Ca^(2+) were detected using Flow cytometry.Fluorescence microscopy was used to observe the autophagy of cells.Knockdown of p53 significantly decreased the expressions of Rap2B protein.Additionally,knockdown of p53 significantly decreased the mRNA levels of PLCε.The knockdown of p53,Rap2B,and PLCεsignificantly decreased the levels of intracellular IP3 and Ca^(2+) and promoted autophagy of U2OS cells.Our results demonstrated that p53-Rap2B-PLCε-IP3 signaling pathway regulated autophagy of U2OS cells.
文摘磷脂酶Cε1(phospholipase C epsilon-1,PLCE1)基因定位于常染色体10q23.32-q24.1,其编码蛋白为磷脂酶Cε1(phospholipase C epsilon-1,PLCε1)。PLCε1蛋白是最新发现的一种磷脂酶C(phospholipase C,PLC)同工酶,在成熟的肾小球足细胞表达,参与肾小球毛细血管袢的形成和正常发育。近来发现PLCE1基因突变可以引起常染色体隐性遗传性肾病综合征。PLCE1基因移码或无义突变可导致婴儿早发性肾病综合征,其病理特征为弥漫性肾小球硬化(diffuse mesangial sclerosis,DMS);而错义突变可导致局灶性节段性肾小球硬化(focal segmental glo-merulosclerosis,FSGS)。但其确切发病情况及机制目前尚不清楚,本文就PLCE1基因及其编码蛋白与激素耐药性肾病综合征之间的关系作一简要综述。
