目的探讨surv iv in在胶质母细胞瘤中的表达及与其凋亡、增殖和微血管密度(M VD)的相关性。方法应用免疫组织化学(SP)法检测42例份胶质母细胞瘤石蜡切片中surv iv in、K i-67和第8因子相关抗原(FⅧRA g)的表达情况,并用末端转移标记法...目的探讨surv iv in在胶质母细胞瘤中的表达及与其凋亡、增殖和微血管密度(M VD)的相关性。方法应用免疫组织化学(SP)法检测42例份胶质母细胞瘤石蜡切片中surv iv in、K i-67和第8因子相关抗原(FⅧRA g)的表达情况,并用末端转移标记法测定凋亡指数(A I)。结果surv iv in在胶质母细胞瘤中的表达阳性率为81.0%,而正常对照组中无1例阳性表达;surv iv in的表达与K i67及M VD呈正相关,而与A I负相关。结论surv iv in在胶质母细胞瘤的发生和发展中可能通过调控细胞的凋亡和增殖起着重要作用,并参与肿瘤血管的生成。可望成为诊断治疗的新靶点。展开更多
目的探讨构建人类富含亮氨酸重复和免疫球蛋白样结构域1(LRIG1)基因过表达慢病毒表达载体并转染胶质瘤细胞系U87细胞的技术方法,为研究LRIG1的功能提供帮助。方法用EcoRⅠ及BamHⅠ双酶切LRIG1基因和plvxDsRed-monomer-n1慢病毒载体,琼...目的探讨构建人类富含亮氨酸重复和免疫球蛋白样结构域1(LRIG1)基因过表达慢病毒表达载体并转染胶质瘤细胞系U87细胞的技术方法,为研究LRIG1的功能提供帮助。方法用EcoRⅠ及BamHⅠ双酶切LRIG1基因和plvxDsRed-monomer-n1慢病毒载体,琼脂糖凝电泳回收LRIG1片段和载体片段;通过T4连接酶将LRIG1基因连接至慢病毒载体上;按Lenti-XHT慢病毒包装试剂盒说明包装慢病毒;用EcoRⅠ及BamHⅠ双酶切法鉴定重组慢病毒载体;然后用plvxDsRed-monomer-n1和plvxDsRed-monomer-n1-3×flagLRIG1分别转染293T细胞和胶质瘤细胞系U87细胞,荧光定量PCR和western blot检测LRIG1 m RNA和蛋白表达。结果 U87细胞感染病毒载体后经嘌呤霉素筛选显示细胞红色荧光较空载体感染细胞减弱;实时PCR结果显示LRIG1 m RNA过表达组较对照明显升高;提取感染后细胞蛋白,Western blot鉴定flag标签蛋白表达成功。结论 LRIG1基因慢病毒表达载体能感染胶质瘤细胞系U87细胞,可使外源基因获得稳定表达。展开更多
The effects of epidermal growth factor （EGF） on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells w...The effects of epidermal growth factor （EGF） on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry （FCM）, and laser scan confocal microscope （LSCM） was used to measure the cytoplasmic free calcium. The results showed that GFAP was diffusedly expressed in GLI5 cells and EGFR was over-expressed. EGF at doses of ≤ 1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas： low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.展开更多
The effects of RNAi-mediated gene silencing of LRlG1 on proliferation and invasion of the human glioma cell line U251-MG and the possible mechanisms were explored in this study. The plasmids pGenesil2-LRIG1-shRNA1 and...The effects of RNAi-mediated gene silencing of LRlG1 on proliferation and invasion of the human glioma cell line U251-MG and the possible mechanisms were explored in this study. The plasmids pGenesil2-LRIG1-shRNA1 and pGenesil2-LRIG1-shRNA2 were transfected into U251-MG glioma cells respectively by using Lipofectamine 2000 and the transfected cells in which the LRIG1 expression was stably suppressed were selected by G418. The cells transfected with negative shRNA served as control. The expression levels of LRIG1 mRNA and protein were measured by qRT-PCR and Western blotting, respectively. The cell cycle was analyzed by flow cytometry. The results showed that LRIG1 mRNA expression was reduced by 70% and 58% and LRIG1 protein expression by 58% and 26% in U251-MG cells transfected with pGenesil2-LRIG1-shRNAl and pGenesil2-LRIG1-shRNA2 relative to the negative shRNA-transfected U251-MG cells. The proliferative capacity of the LRIG1 specific siRNA-transfected cells was stronger than that of control cells. Cell cycle analysis showed that silencing LRIG1 significantly increased the percentage of S phase cells and the proliferation index (P<0.01). Moreover, silencing LRIG1 could promote the invasion of U251-MG cells (P<0.05). These findings suggested that LRIG1-targeting siRNA can exert a dramatically inhibitory effect on RNA transcription and protein expression of LRIG1, and LRIG1 down-regulation could promote the proliferation of U251-MG cells, arrest U251-MG cells in S phase, and enhance the invasion of U251-MG cells.展开更多
文摘目的探讨surv iv in在胶质母细胞瘤中的表达及与其凋亡、增殖和微血管密度(M VD)的相关性。方法应用免疫组织化学(SP)法检测42例份胶质母细胞瘤石蜡切片中surv iv in、K i-67和第8因子相关抗原(FⅧRA g)的表达情况,并用末端转移标记法测定凋亡指数(A I)。结果surv iv in在胶质母细胞瘤中的表达阳性率为81.0%,而正常对照组中无1例阳性表达;surv iv in的表达与K i67及M VD呈正相关,而与A I负相关。结论surv iv in在胶质母细胞瘤的发生和发展中可能通过调控细胞的凋亡和增殖起着重要作用,并参与肿瘤血管的生成。可望成为诊断治疗的新靶点。
文摘目的探讨构建人类富含亮氨酸重复和免疫球蛋白样结构域1(LRIG1)基因过表达慢病毒表达载体并转染胶质瘤细胞系U87细胞的技术方法,为研究LRIG1的功能提供帮助。方法用EcoRⅠ及BamHⅠ双酶切LRIG1基因和plvxDsRed-monomer-n1慢病毒载体,琼脂糖凝电泳回收LRIG1片段和载体片段;通过T4连接酶将LRIG1基因连接至慢病毒载体上;按Lenti-XHT慢病毒包装试剂盒说明包装慢病毒;用EcoRⅠ及BamHⅠ双酶切法鉴定重组慢病毒载体;然后用plvxDsRed-monomer-n1和plvxDsRed-monomer-n1-3×flagLRIG1分别转染293T细胞和胶质瘤细胞系U87细胞,荧光定量PCR和western blot检测LRIG1 m RNA和蛋白表达。结果 U87细胞感染病毒载体后经嘌呤霉素筛选显示细胞红色荧光较空载体感染细胞减弱;实时PCR结果显示LRIG1 m RNA过表达组较对照明显升高;提取感染后细胞蛋白,Western blot鉴定flag标签蛋白表达成功。结论 LRIG1基因慢病毒表达载体能感染胶质瘤细胞系U87细胞,可使外源基因获得稳定表达。
文摘The effects of epidermal growth factor （EGF） on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry （FCM）, and laser scan confocal microscope （LSCM） was used to measure the cytoplasmic free calcium. The results showed that GFAP was diffusedly expressed in GLI5 cells and EGFR was over-expressed. EGF at doses of ≤ 1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas： low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.
基金supported by grants from the National Natural Science Foundation of China(No.30500521and No.81001116)
文摘The effects of RNAi-mediated gene silencing of LRlG1 on proliferation and invasion of the human glioma cell line U251-MG and the possible mechanisms were explored in this study. The plasmids pGenesil2-LRIG1-shRNA1 and pGenesil2-LRIG1-shRNA2 were transfected into U251-MG glioma cells respectively by using Lipofectamine 2000 and the transfected cells in which the LRIG1 expression was stably suppressed were selected by G418. The cells transfected with negative shRNA served as control. The expression levels of LRIG1 mRNA and protein were measured by qRT-PCR and Western blotting, respectively. The cell cycle was analyzed by flow cytometry. The results showed that LRIG1 mRNA expression was reduced by 70% and 58% and LRIG1 protein expression by 58% and 26% in U251-MG cells transfected with pGenesil2-LRIG1-shRNAl and pGenesil2-LRIG1-shRNA2 relative to the negative shRNA-transfected U251-MG cells. The proliferative capacity of the LRIG1 specific siRNA-transfected cells was stronger than that of control cells. Cell cycle analysis showed that silencing LRIG1 significantly increased the percentage of S phase cells and the proliferation index (P<0.01). Moreover, silencing LRIG1 could promote the invasion of U251-MG cells (P<0.05). These findings suggested that LRIG1-targeting siRNA can exert a dramatically inhibitory effect on RNA transcription and protein expression of LRIG1, and LRIG1 down-regulation could promote the proliferation of U251-MG cells, arrest U251-MG cells in S phase, and enhance the invasion of U251-MG cells.
