死亡受体5胞外区域的重组、表达及活性鉴定

目的构建死亡受体5(deathreceptor5,DR5)胞外区域(eDR5)的表达载体,表达纯化重组蛋白并鉴定其生物特性。方法通过重叠PCR获得DR5胞外段编码序列,构建pET-22b(+)/DR5表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,Ni2+柱亲和纯化,SDS-PAGE、直接ELISA鉴定纯化产物的纯度和特异性,用MTT法检测eDR5蛋白阻断DR5单克隆抗体FMU1.5和TRAIL诱导人胶质瘤细胞株U343(高表达DR5)、U373(低表达DR5)细胞凋亡的作用。结果获得了DR5胞外段编码序列,目的蛋白在上清及包涵体中都有表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上,蛋白产量达9mg/ml。ELISA结果表明所纯化蛋白为eDR5。eDR5蛋白可部分阻断FMU1.5和TRAIL诱导人胶质瘤细胞株U343细胞凋亡的作用,其阻断率与DR5表达相关。结论死亡受体5胞外段基因的成功重组、表达及纯化,为进一步的功能研究奠定了基础

Regulation of Cell Cycle of Glioma C6 Cells by Regulator of G Protein Signaling 16

目的:探讨G蛋白调节子 16(RGS16)对胶质瘤C6细胞周期的影响。方法:利用脂质体介导法将RGS16基因导入C6细胞中 ,在倒置显微镜下观察细胞形态变化和贴壁生长情况 ;免疫细胞化学法检测转染前后RGS16蛋白的表达情况 ;流式细胞仪检测转染pCMV 5 RGS16和 pCMV 5质粒后每隔 12h后的细胞周期变化。 结果:转染pCMV 5 RGS16质粒 2 4h后 3 0 .0 %细胞贴壁性降低 ,突起收缩 ,细胞变圆 ,72h之后细胞又恢复正常 ;RGS16蛋白的表达呈时相性 ,3 6h时表达率最高 (阳性率为13 .0 % ) ,72h表达终止 ;C6细胞的各期细胞比例变化与RGS16蛋白表达对应 ,在 3 6h时G1期比例从转染前的 70 .5 %降低到60 .2 % ,S期比例从 2 0 .9%增加到 3 4.9% ;在 48h时G1期增加到 76.2 % ,S期减少到 11.4% ;72h各期恢复到正常比例。对照组细胞转染前后形态变化不明显 ,RGS16蛋白表达阴性 ,细胞周期变化不明显。结论:RGS16能促进C6细胞周期的运行. 【英文摘要】 Objective To study the effect of regulator of G protein signaling 16(RGS16) on the cell cycle of glioma C6 cell.Methods pCMV5 RGS16 was transfected into C6 cells by lipofectin.The morphological and adhesive changes of the cells were observed under an inverted microscope.Expression of RGS16 was examined by immunocytochemical method both before and after the transfection.Flow Cytometry was adopted to measure the fraction number changes of the cell cycle phase every 12 h.Results 24 hours after the transfec..

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

Rat glioma C6 cell apoptosis induced by UV radiation via p38-MAPK

目的 :探讨 p38MAPK在紫外线损伤刺激细胞中的特异性信号转导作用 .方法 :流式细胞仪检测紫外线照射 30min后 1,2及 4h的C6细胞周期变化和是否有凋亡发生 ;应用免疫细胞化学技术观察紫外线刺激前后 p38MAPK在C6细胞中的表达强度和分布特征 .结果 :细胞周期结果显示 1,2和 4h后G1期细胞数分数各增多 0 .12 ,0 .2 1和 0 .19,而S期细胞数分数减少 0 .10 ,0 .14和 0 .15 ;各组的凋亡率分别是12 % ,4 9%和 34% ;未受刺激的细胞中 ,p38MAPK在胞质和胞核表达较弱 ;紫外线损伤作用 2h后 ,细胞核区的染色强度即明显增强 ,而胞质区域的染色强度相对降低 .结论 :C6细胞受紫外线损伤后可通过 p38MAPK通路发生凋亡. 【英文摘要】 AIM: To study the signal transduction of p38 mitogen activated protein kinase in rat glioma C6 cells after the stimulation of UV radiation. METHODS: Flow cytometry was applied to measure the fraction number changes in the cell cycle phase and to detect whether UV could induce apoptosis of C6 cells. The level and distribution of p38MAPK expression was examined by immunocytochemical method both before and after the UV radiation. RESULTS: Flow cytometry indicated that the numbers of G1 phase fraction of 1,...高等学校骨干教师计划资助 ;; 留学归国人员科研启动基金([1 999] 747号

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

p38MAPK与HSP70关系的初步研究

高等学校骨干教师资助计划 ;; 留学归国人员科研启动基金([1999] 747号

Regulation of Cell Cycle of Glioma C6 Cells by Regulator of G Protein Signaling 16

目的　探讨G蛋白调节子 16(RGS16)对胶质瘤C6细胞周期的影响。方法　利用脂质体介导法将RGS16基因导入C6细胞中 ,在倒置显微镜下观察细胞形态变化和贴壁生长情况 ;免疫细胞化学法检测转染前后RGS16蛋白的表达情况 ;流式细胞仪检测转染pCMV 5 RGS16和 pCMV 5质粒后每隔 12h后的细胞周期变化。 结果　转染pCMV 5 RGS16质粒 2 4h后 3 0 .0 %细胞贴壁性降低 ,突起收缩 ,细胞变圆 ,72h之后细胞又恢复正常 ;RGS16蛋白的表达呈时相性 ,3 6h时表达率最高 (阳性率为13 .0 % ) ,72h表达终止 ;C6细胞的各期细胞比例变化与RGS16蛋白表达对应 ,在 3 6h时G1期比例从转染前的 70 .5 %降低到60 .2 % ,S期比例从 2 0 .9%增加到 3 4.9% ;在 48h时G1期增加到 76.2 % ,S期减少到 11.4% ;72h各期恢复到正常比例。对照组细胞转染前后形态变化不明显 ,RGS16蛋白表达阴性 ,细胞周期变化不明显。结论　RGS16能促进C6细胞周期的运行 【英文摘要】 Objective To study the effect of regulator of G protein signaling 16(RGS16) on the cell cycle of glioma C6 cell.Methods pCMV5 RGS16 was transfected into C6 cells by lipofectin.The morphological and adhesive changes of the cells were observed under an inverted microscope.Expression of RGS16 was examined by immunocytochemical method both before and after the transfection.Flow Cytometry was adopted to measure the fraction number changes of the cell cycle phase every 12 h.Results 24 hours after the transfec..

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 【英文摘要】 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

Promotion of glioma C6 cells proliferation by overexpressed RGS16

目的　探讨 G蛋白调节子 16 (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RGS16基因导入 C6细胞中 ,在倒置显微镜下观察细胞形态变化和贴壁情况 ;3H- Td R法检测 C6细胞在转染不同梯度p CMV5 - RGS16和 p CMV 5质粒后的增殖情况 ;免疫细胞化学法检测转染前后 RGS16蛋白的表达情况 ;流式细胞仪检测转染 p CMV5 - RGS16和 p CMV5质粒 36 h后细胞周期变化和细胞是否有凋亡发生 .结果　转染 p CMV5 - RGS16质粒 2 4 h后 30 %细胞贴壁性降低 ,突起收缩 ,细胞变圆 ;RGS16蛋白表达阳性 ;3H- Td R法检测显示 C6细胞增殖速度与转染p CMV5 - RGS16的量呈正相关 ;细胞周期结果显示 G1期细胞百分数减少 10 % ,而 S期细胞百分数增多 14 % ;未发现RGS16与凋亡有直接关系 .结论　 RGS16可能促进 C6细胞的增殖 . 【英文摘要】 s: AIM To study the effect of RGS16 on the biological characteristics of glioma C6 cells. METHODS pCMV5 RGS16 was transfected into C6 cells by lipofectin. The morphological and adhesive changes of the cells were observed under an inverted microscope. Proliferation of C6 cells was measured by 3H thymidine ( 3H TdR) assay after gradient transfections of pCMV5 RGS16 and pCMV5. Expression of RGS16 was examined by immunocytochemical method both before and after the transfection. Flow cytometry ...高等学校骨干教师计划资

A 1, 2, 3 - triazole class starch derivative and its preparation method and application

本发明涉及日化领域及医药行业，具体是一种1, 2, 3‑三氮唑类淀粉衍生物及其制备方法和应用。1, 2, 3‑三氮唑类淀粉衍生物结构式如式(1)所示，其中，R为含有不同活性基团的等链长取代基；平均聚合度n取值范围是5‑12000。本发明反应高效，易于推广，所需设备及原料易得。研究表明合成的1, 2, 3‑三氮唑类淀粉衍生物水溶性好，具有极好的抑菌活性，增强了淀粉的生物活性，扩大了淀粉的应用范围，可以广泛应用于日化及医药领域