非小细胞肺癌细胞株HCC827克隆异质性及其与吉非替尼敏感性的关系  

Clone heterogeneity of non-small-cell lung cancer cell line HCC827 and its relationship with gefitinib sensitivity

作　　者：罗凯 黎谢梦丹 董静 王倩 LUO Kai;LI Xiemengdan;DONG Jing;WANG Qian(Cancer Center of Guangzhou Medical University/Cancer Institute of Guangzhou Medical University,Guangzhou 510095,China)

机构地区：[1]广州医科大学附属肿瘤医院/广州医科大学肿瘤研究所,广东广州510095

出　　处：《山东医药》2020年第33期1-5,共5页Shandong Medical Journal

基　　金：广东省医学科学技术研究基金项目(B2019063);广州市卫生和计划生育科技项目(20191A011099)。

摘　　要：目的观察非小细胞肺癌(NSCLC)细胞株HCC827的克隆异质性,分析其与吉非替尼敏感性的关系。方法取对数生长期HCC827细胞,利用极限稀释法获得单克隆化HCC827子代细胞株,分为A、B、C、D、E、F组;另取对数生长期HCC827细胞,记为亲代细胞组。采用平板克隆形成实验观察各组细胞平板克隆形成数,采用细胞生长曲线实验观察各组细胞增殖倍数,观察细胞增殖能力异质性;采用细胞药敏实验观察各组细胞对吉非替尼敏感异质性;采用Sanger直接测序法检测各组细胞EGFR基因,采用荧光原位杂交实验检测各组细胞MET、HER2基因,观察各组细胞遗传异质性;采用细胞克隆混合实验观察吉非替尼对细胞异质性的动态影响。结果B组克隆形成数高于亲代细胞组(P<0.01),A、D、E组克隆形成数低于亲代细胞组(P均<0.05)。B、F组细胞培养48、72、96、120 h时的增殖倍数与亲代细胞组相比,P均<0.05;C、D组细胞培养72、96、120 h时的增殖倍数与亲代细胞组相比,P均<0.05;其中B组细胞增殖倍数最高,D组细胞增殖倍数最低。B组细胞为对吉非替尼最敏感的细胞组,其余各组细胞对吉非替尼的IC50值与亲代细胞组相比,P均<0.05,其中D组细胞为对吉非替尼IC50值最高的细胞组。亲代细胞组与A^F组细胞均为EGFR基因19 Del突变。D组细胞HER2基因扩增为阳性、MET基因扩增为阴性,而亲代细胞组和A、B、C、E、F组细胞HER2基因和MET基因扩增均为阴性。在吉非替尼压力下,吉非替尼敏感B克隆细胞和耐药D克隆细胞等比例混合培养2周后耐药细胞获得绝对生长优势,B克隆细胞与亲代细胞等比例混合培养2周后少量残留细胞中亲代细胞存在相对优势,D克隆细胞与亲代细胞等比例混合培养2周后两细胞均势生长。结论HCC827细胞在增殖能力、药物敏感性等表型层面和驱动基因等遗传背景层面均存在克隆异质性,并且其克隆异质性与吉非替尼敏Objective To explore the heterogeneity of non-small-cell lung cancer(NSCLC)cell line HCC827,and to analyze its relationship with gefitinib sensitivity.Methods We took HCC827 cells in the logarithmic growth phase as the research object,used the limiting dilution method to obtain the monoclonal HCC827 progeny cells,and divided them into groups A,B,C,D,E,and F.In addition,HCC827 cells in the logarithmic growth phase were used as the parental cell group.The plate clone formation experiment was used to observe the number of plate colonies formed in cells of each group.Cell proliferation multiples of each group were detected through cell growth curve experiment and the heterogeneity of cell proliferation ability was observed.The cell drug sensitivity test was used to observe the heterogeneity of the sensitivity of cells to gefitinib in each group.Sanger sequencing method was used to detect the EGFR gene of each group.The fluorescence in situ hybridization was used to detect the MET,HER2 genes of each group,and the genetic heterogeneity of each group was observed.Cell clone mixing experiment was used to observe the dynamic effect of gefitinib on cell heterogeneity.Results The number of clone formation in the group B was higher than that of the parental cell group(P<0.01),and the number of clone formation in the groups A,D,and E was lower than that of the parental cell group(all P<0.05).Significant difference was found in the proliferation folds of cells between groups A-F and parental cell group at 48,72,96,and120 h(all P<0.05);the the proliferation fold of group B was the highest,and the proliferation fold of group D was the lowest.The cells of group B were the most sensitive cells to gefitinib.Compared with the parent cell group,the IC50 values of cells to gefitinib in the other groups were significantly different(all P<0.05),and group D was the cell group with the highest IC50 value for gefitinib.Both the parental cell group and the groups A-F had EGFR gene 19 Del mutation.The amplification of HER2 gene in the group

关 键 词：非小细胞肺癌 克隆异质性 遗传异质性 吉非替尼 药物耐受 细胞实验 

分 类 号：R73-3[医药卫生—肿瘤]