Effect of Exogenous RGS16 Gene Transfection on Growth of Glioma Cell Line C6

背景与目的:有研究表明野生型p53可以诱导RGS16表达,而RGS16可能与胶质瘤的发生有关。本研究旨在探讨RGS16基因转染对大鼠胶质瘤C6细胞生长的影响。方法:构建真核表达载体pIRES2-EGFP-RGS16,脂质体法转染C6细胞,经G418筛选后荧光显微镜观察细胞中增强型绿色荧光蛋白(enhancedgreenfluorscentprotein,EGFP)的表达,RT-PCR证实RGS16的mRNA表达,免疫细胞化学方法检测细胞中RGS16蛋白表达。最后利用流式细胞仪、生长曲线、平板克隆形成等方法研究RGS16基因对胶质瘤细胞周期、生长及增殖的影响。结果:成功构建了稳定表达RGS16和表达空载体的细胞系C6-RGS16、C6-GFP。C6-RGS16、C6-GFP和C6经流式细胞仪检测S期的细胞比例分别为28.5%、18.9%和14.3%(P<0.05);生长曲线表明C6-RGS16生长的速度明显快于C6-GFP及C6,但其平板克隆形成率分别为12%、25%和25%(P<0.05)。结论:RGS16促进C6细胞周期从G1期向S期过渡,加快细胞生长速度,但是并不促进细胞克隆形成。 【英文摘要】 BACKGROUND & OBJECTIVE: Wild type p53 could induceRGS16 mRNA expression, and RGS16 protein is related with carcinogenesisof glioma. This study was designed to investigate the effects of exogenousRGS16 gene transfection on the growth of rat glioma cell line C6.METHODS : A eukaryotic expression plasmid pIRES2-EGFP-RGS16 wasconstructed, and transfected into C6 cells. The cells were selected by G418.The expression of enhanced green fluorescent protein (EGFP) was observedunder fluorescent microscope, and the exp...军队医药卫生科研基金项目(No.02ma04)~

Detection of point mutations of Axin gene and its expression in gliomas

目的　检测胶质瘤中Axin基因的点突变及其表达情况 ,初步探讨Axin与胶质瘤发生的关系。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序方法检测Axin基因外显子 8,9及 10在 2 8例胶质瘤中的突变情况 ;同时对上述胶质瘤及正常脑组织进行免疫组化染色。结果　在 2 8例胶质瘤组织中Axin的第 10个外显子共有 6例样本 (2 1.4 % ) 3处发生了错义突变 ;3例 3处发生了同义突变 ;2 8例胶质瘤中 8例 (2 8.6 % )Axin表达阳性 ,正常脑组织中神经元表达阳性 ,神经胶质细胞表达阴性 ,检测到突变的样本中 1例表达阳性。结论　Axin基因的点突变可能参与胶质瘤的发生 【英文摘要】 Objective To detect the point mutations of Axin gene and its expression in glioma and explore the relationship between Axin gene and the occurrence of human glioma.Methods The point mutations of exon 8,9,10 of Axin gene were analyzed in 28 cases of glioma by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) analysis, silver staining and DNA sequencing. Immunohistochemistry was used to detect the Axin expression in these cases and normal brain tissues.Results Three missense poi...高等学校骨干教师计划资助项目;; 归国留学人员科研启动基金资助项目 (1 999747

Effects of transfected HSP70 on p38MAPK signal pathway

目的:探讨热休克蛋白 (HSP70 )在人胶质瘤细胞BT 32 5 p38MAPK信号通路中的作用。方法:用脂质体介导法将hsp70基因导入人胶质瘤细胞BT 32 5中 ,倒置显微镜观察转染细胞的形态学及粘附性变化 ,紫外线照射 30min后 ,采用免疫组化和Western blot方法测定转染前后HSP70的表达水平及照射前后 p38MAPK表达情况。结果:免疫组化和Western blot证实hsp70基因成功转染入BT 32 5中 ,转染细胞受到紫外线照射后 p38MAPK表达减弱。结论:体外转染hsp70基因可抑制紫外线照射后BT 32 5细胞 p38MAPK的表达. Objective To study the role of HSP70 in p38MAPK signal transduction of human glioma cells BT 325.Methods pBBS212 hsp70 gene was transfected into BT 325 cells by lipofectin. The morphological and adhesive changes of the cells were observed under an inverted microscope. The level of HSP70 was measured by immunohistochemistry. Then the transfected cells were put into ultraviolet (UV) for 30 minitues, and expression of p38MAPK and HSP70 were examined by immunohistochemistry and Western blot methods bo...国家自然科学基金资助项目 (30 1 0 0 2 1 8);; 高等学校骨干教师资助计划 (2 0 0 0 - 65 - 66);; 留学归国人员科研启动基金资助项目 (1 999- 747

Expression and relationship of RGS16 and p53 in human glioma

目的研究G蛋白信号调节子16(RGS16)与p53在人胶质瘤中表达及其相关性。方法利用免疫组织化学链霉亲合素生物素过氧化物酶复合物(SABC)法检测42例胶质瘤标本中RGS16与p53的表达。结果RGS16在10例胶质瘤旁正常脑组织有8例神经元阳性但胶质细胞阴性、42例胶质瘤中37例肿瘤细胞阳性,二者差异显著(P0.05);其中,RGS16与p53共同阳性表达11例,共同阴性表达1例,Kappa检验二者表达呈负相一致(P<0.05)。结论RGS16在胶质瘤中高表达而与病理分级无关,推测RGS16可能在胶质瘤发生中起作用。另外,胶质瘤中RGS16与p53表达呈负相关。 【英文摘要】 Objective To study the expression of RGS16 in human glioma and its relationship with p53. Methods The expression of RGS16 and p53 in protein level was studied by immunohistochemistry strept avidin-biotion-peroxidase-complex (SABC) method in 42 samples.Results In 10 normal brain tissues beside the human glioma, 8 cases expressed RGS16 in the neurons,but none in gliocytes; in 42 human gliomas, 37 cases expressed RGS16 in the glioma cells, and the difference between the normal tissue and the glioma was observe...军队医药卫生科研基金资助项目(02ma04);; 国家杰出青年自然科学基金资助项目(30125012);; 高等学校骨干教师及归国留学人员科研启动基金资助项目([1999]747

Construction of the eukaryotic expression vector pIRES2-EGFP-Axin and its expression in glioma cells

目的:构建真核表达载体pIRES2EGFPAxin,并在神经胶质瘤细胞系C6中进行表达。方法:用PCR的方法扩增Axin基因,构建真核表达载体pIRES2EGFPAxin,经NheI及SalI双酶切鉴定并测序。通过脂质体法转染C6细胞,用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用免疫组化染色的方法检测细胞中Axin的表达。结果:构建了真核表达载体pIRES2EGFPAxin,用脂质体法转染神经胶质瘤细胞C6后,经荧光显微镜和免疫组化染色法检测,可见细胞内有EGFP及Axin的表达。结论:成功地构建真核表达载体pIRES2EGFPAxin,并在神经胶质瘤细胞C6中表达,为研究Axin对肿瘤的生物学作用以及Axin在肿瘤基因治疗中的应用奠定了基础。国家杰出青年自然科学基金项目(No.30125012);; 军队医药卫生科研基金项目(No.02ma04);; 高等学校骨干教师及归国留学人员科研启动基金(No.1999747

Impact of p38MAPK and RGS16 to the apoptosis and cell cycle of the glioma C6 cells

目的　探讨p38MAPK和RGS16对大鼠胶质瘤C6细胞的生物学特性的影响 .方法　利用脂质体介导法将p38MAPK和RGS16基因分别或共转染导入C6细胞中 ;用p38MAPK抑制剂SB 2 0 2 190处理处于对数生长期的转染和未转染 p38MAPK的C6细胞 ,2 4～ 36h后在倒置显微镜下观察细胞形态变化和贴壁情况 ;免疫细胞化学法检测转染前后p38MAPK和RGS16蛋白的表达情况 ;流式细胞仪检测细胞周期变化和细胞是否有凋亡发生 .结果　转染 pCMV5 p38和 /或pCMV5 RGS16质粒 36h后 30 %细胞贴壁性降低 ,突起收缩 ,细胞变圆 ;p38MAPK和RGS16蛋白均表达阳性 ;转染pCMV5 p38组出现 33.8%的凋亡峰 ,细胞周期结果显示G1期细胞百分数增加 17% ,而S期细胞百分数减少 14 % ;转染pCMV5 RGS16组无凋亡发生 ,细胞周期结果显示G1期细胞百分数减少 10 % ,而S期细胞百分数增加 14 % ;共转染pCMV5 P38和pCMV5 RGS16组未出现的凋亡峰 ,细胞周期结果显示G1期和S期细胞百分数变化与未处理组之间没有明... 【英文摘要】 AIM To study the effects of p38MAPK and RGS16 on the biological characteristics of glioma C6 cells. METHODS pCMV5 p38 and pCMV5 RGS16 were respectively or jointly transfected into C6 cells by lipofectin. SB 202190 was used to treat the transfected and nontransfected pCMV5 p38 C6 cells. The morphological and adhesive changes of the cells were observed under an inverted microscope. Expression of p38 and RGS16 was examined by immunocytochemical method both before and after the transfection. Flow Cytometr...高等学校骨干教师资助计划项目 ;; 留学归国人员科研启动基金项目 ([1 999] 747号

Construction of the eukaryotic expression vector pIRES2-EGFP-Axin and its expression in glioma cells

目的:构建真核表达载体pIRES2EGFPAxin,并在神经胶质瘤细胞系C6中进行表达。方法:用PCR的方法扩增Axin基因,构建真核表达载体pIRES2EGFPAxin,经NheI及SalI双酶切鉴定并测序。通过脂质体法转染C6细胞,用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用免疫组化染色的方法检测细胞中Axin的表达。结果:构建了真核表达载体pIRES2EGFPAxin,用脂质体法转染神经胶质瘤细胞C6后,经荧光显微镜和免疫组化染色法检测,可见细胞内有EGFP及Axin的表达。结论:成功地构建真核表达载体pIRES2EGFPAxin,并在神经胶质瘤细胞C6中表达,为研究Axin对肿瘤的生物学作用以及Axin在肿瘤基因治疗中的应用奠定了基础。 【英文摘要】 AIM: To construct the eukaryotic expression vector pIRES2-EGFP-Axin, and to express Axin in C6 glioma cells. METHODS: The Axin gene was amplified by PCR using pCMV5-HA-Axin as a template, and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-Axin was constructed by introducing Axin DNA fragment into the sites of Nhe I and Sal I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the...国家杰出青年自然科学基金项目(No.30125012);; 军队医药卫生科研基金项目(No.02ma04);; 高等学校骨干教师及归国留学人员科研启动基金(No.1999747

Detection of point mutations of Axin gene and its expression in gliomas

目的　检测胶质瘤中Axin基因的点突变及其表达情况 ,初步探讨Axin与胶质瘤发生的关系。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序方法检测Axin基因外显子 8,9及 10在 2 8例胶质瘤中的突变情况 ;同时对上述胶质瘤及正常脑组织进行免疫组化染色。结果　在 2 8例胶质瘤组织中Axin的第 10个外显子共有 6例样本 (2 1.4 % ) 3处发生了错义突变 ;3例 3处发生了同义突变 ;2 8例胶质瘤中 8例 (2 8.6 % )Axin表达阳性 ,正常脑组织中神经元表达阳性 ,神经胶质细胞表达阴性 ,检测到突变的样本中 1例表达阳性。结论　Axin基因的点突变可能参与胶质瘤的发生高等学校骨干教师计划资助项目;; 归国留学人员科研启动基金资助项目 (1 99974

Effect of Exogenous RGS16 Gene Transfection on Growth of Glioma Cell Line C6

背景与目的:有研究表明野生型p53可以诱导RGS16表达,而RGS16可能与胶质瘤的发生有关。本研究旨在探讨RGS16基因转染对大鼠胶质瘤C6细胞生长的影响。方法:构建真核表达载体pIRES2-EGFP-RGS16,脂质体法转染C6细胞,经G418筛选后荧光显微镜观察细胞中增强型绿色荧光蛋白(enhancedgreenfluorscentprotein,EGFP)的表达,RT-PCR证实RGS16的mRNA表达,免疫细胞化学方法检测细胞中RGS16蛋白表达。最后利用流式细胞仪、生长曲线、平板克隆形成等方法研究RGS16基因对胶质瘤细胞周期、生长及增殖的影响。结果:成功构建了稳定表达RGS16和表达空载体的细胞系C6-RGS16、C6-GFP。C6-RGS16、C6-GFP和C6经流式细胞仪检测S期的细胞比例分别为28.5%、18.9%和14.3%(P<0.05);生长曲线表明C6-RGS16生长的速度明显快于C6-GFP及C6,但其平板克隆形成率分别为12%、25%和25%(P<0.05)。结论:RGS16促进C6细胞周期从G1期向S期过渡,加快细胞生长速度,但是并不促进细胞克隆形成。军队医药卫生科研基金项目(No.02ma04)~

Effect of Exogenous RGS16 Gene Transfection on Growth of Glioma Cell Line C6

背景与目的:有研究表明野生型p53可以诱导RGS16表达,而RGS16可能与胶质瘤的发生有关。本研究旨在探讨RGS16基因转染对大鼠胶质瘤C6细胞生长的影响。方法:构建真核表达载体pIRES2-EGFP-RGS16,脂质体法转染C6细胞,经G418筛选后荧光显微镜观察细胞中增强型绿色荧光蛋白(enhancedgreenfluorscentprotein,EGFP)的表达,RT-PCR证实RGS16的mRNA表达,免疫细胞化学方法检测细胞中RGS16蛋白表达。最后利用流式细胞仪、生长曲线、平板克隆形成等方法研究RGS16基因对胶质瘤细胞周期、生长及增殖的影响。结果:成功构建了稳定表达RGS16和表达空载体的细胞系C6-RGS16、C6-GFP。C6-RGS16、C6-GFP和C6经流式细胞仪检测S期的细胞比例分别为28.5%、18.9%和14.3%(P<0.05);生长曲线表明C6-RGS16生长的速度明显快于C6-GFP及C6,但其平板克隆形成率分别为12%、25%和25%(P<0.05)。结论:RGS16促进C6细胞周期从G1期向S期过渡,加快细胞生长速度,但是并不促进细胞克隆形成。 【英文摘要】 BACKGROUND & OBJECTIVE: Wild type p53 could induceRGS16 mRNA expression, and RGS16 protein is related with carcinogenesisof glioma. This study was designed to investigate the effects of exogenousRGS16 gene transfection on the growth of rat glioma cell line C6.METHODS : A eukaryotic expression plasmid pIRES2-EGFP-RGS16 wasconstructed, and transfected into C6 cells. The cells were selected by G418.The expression of enhanced green fluorescent protein (EGFP) was observedunder fluorescent microscope, and the exp...军队医药卫生科研基金项目(No.02ma04)~