脑胶质瘤U251多倍体巨细胞的构建及其对替莫唑胺的耐药性研究  

Establishment of the U251 glioma polyploidy giant cancer cells and study of their characteristic of temozolomide resistance

作　　者：米蕊芳[1] 季佳宇 王启俨 方胜 田毅夫 刘福生[1,2] Mi Ruifang;Ji Jiayu;Wang Qiyan;Fang Sheng;Tian Yifu;Liu Fusheng(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;Department of Neurosurgery,Beijing Tiantan Hospital,Capital Medical University,Beijing 100070,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,100070 [2]首都医科大学附属北京天坛医院神经外科,100070

出　　处：《中华神经外科杂志》2021年第2期193-197,共5页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(81672478);北京市自然科学基金(7182026)。

摘　　要：目的探讨脑胶质瘤U251多倍体巨细胞的体外构建方法并研究其对替莫唑胺的耐药性。方法通过慢病毒感染实验观察带有不同荧光标记的人脑胶质瘤U251细胞;采用植物血凝素-聚乙二醇融合法进行细胞融合;通过流式细胞分选实验检测体外携带双色荧光标记的胶质瘤多倍体巨细胞;采用碘化丙啶染色方法测定U251融合克隆细胞的DNA含量;通过CCK-8实验评价细胞的增殖率以及加入替莫唑胺后细胞的存活率。结果慢病毒感染实验成功获得分别带有绿色和红色荧光标记的胶质瘤U251细胞;细胞融合后显微镜下可见携带双色荧光的胶质瘤融合细胞,并且与聚乙二醇细胞融合法比较,植物血凝素-聚乙二醇细胞融合法的细胞融合率明显提高(自<1%提高至>5%)。流式细胞分选获得胶质瘤U251融合细胞单克隆株。DNA含量的检测结果显示,U251融合克隆细胞的DNA含量自之前的2N/4N提高至4N/8N。CCK-8实验结果显示,U251细胞接种后1、2、3、4及5 d的细胞增殖率分别为(99.5±2.6)%、(236.7±4.7)%、(348.9±8.8)%、(656.5±18.5)%及(715.7±48.2)%;而U251-C1多倍体巨细胞的细胞增殖率分别为(100.6±2.1)%、(114.5±1.3)%、(138.4±1.7)%、(316.7±23.1)%及(347.7±13.1)%,U251-C2多倍体巨细胞的细胞增殖率分别为(100.3±1.2)%、(114.7±1.2)%、(186.4±0.6)%、(307.1±9.5)%及(432.9±37.7)%,相较于胶质瘤U251细胞,U251多倍体巨细胞的不同克隆株(U251-C1、U251-C2)的增殖速度均明显减慢(均P<0.05)。替莫唑胺作用下,U251细胞和U251多倍体巨细胞(U251-C1、U251-C2)的细胞存活率均下降(均P<0.01),而与U251细胞组比较,不同U251多倍体巨细胞株(U251-C1、U251-C2)的细胞存活率均更高(均P<0.01)。结论植物血凝素-聚乙二醇细胞融合法可在体外成功构建脑胶质瘤U251多倍体巨细胞;胶质瘤U251多倍体巨细胞可能对替莫唑胺的耐药性发挥重要作用。Objective To establish the U251 glioma polyploidy giant cells(GPGCs)and to study their characteristic of temozolomide(TMZ)resistance in vitro.Methods We obtained the U251 glioma cells canning EGFP and mCherry glioma markers respectively by lentivirus infection,acquired the GPGCs by phytohemagglutinin-polyethylene glycol(PHA-PEG)fusion methods,and obtained the fusion cell clones by fluorescence activated cell sorting(FACS).DNA content was detected by PI staining and flow cytometry analysis.We evaluated the cell proliferation rate and cell survival rate under TMZ(temozolomide)by CCK-8 method.Results U251 glioma cells carrying EGFP or mCherry marker were firstly obtained.The fusion rate was significantly increased(from <1% to >5%)by the PHA-PEG fusion method compared to PEG fusion method.Then the U251 GPGCs were successfully acquired.DNA content of the U251 GPGCs was transfected from 2 N/4 N to 4 N/8 N.The proliferation rates of the U251 cells were(99.5±2.6)%,(236.7±4.7)%,(348.9±8.8)%,(656.5±18.5)% and(715.7±48.2)%respectively at 1 d,2 d,3 d,4 d and 5 d after cell implantation,while the proliferation rates of the U251-C1 cells were(100.6±2.1)%,(114.5±1.3)%,(138.4±1.7)%,(316.7±23.1)% and(347.7±13.1)%,and the proliferation rates of the U251-C2 cells were(100.3±1.2)%,(114.7±1.2)%,(186.4±0.6)%,(307.1±9.5)% and(432.9±37.7)% respectively at 1 d,2 c1,3 c1,4 d and 5 d after cell implantation.The proliferation rate of the U251 GPGCs was significantly decreased compared to U251 cells(all P<0.05).The survival rate of the TMZ group was significantly decreased compared to control group(all P<0.01),while the survival rate of the U251 PGCCs group was significantly infreased compared to U251 group(all P<0.01).Conclusions The U251 GPGCs model could he successfully established using the phytohemagglutinin-polyethylene glycol(PHA-PEG)fusion methods.GPGCs may play important roles in the TMZ resistance of gliomas.

关 键 词：神经胶质瘤 细胞融合 多倍体巨细胞 替莫唑胺 耐药性 

分 类 号：R739.41[医药卫生—肿瘤]