摘要
目的比较初发和复发胶质母细胞瘤(GBM)标本、体外培养的原代胶质瘤干细胞及患者原代肿瘤组织来源的肿瘤移植模型(PDX)的分子遗传学特征。方法(1)取同一患者手术切除的初发及复发GBM标本,免疫组化染色检测标本胶质纤维酸性蛋白(GFAP)、巢蛋白、Ki-67的表达;基因检测分析标本O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)基因甲基化、异柠檬酸脱氢酶(IDH)基因突变及表皮生长因子受体(EGFR)基因扩增情况。(2)体外原代培养初发及复发GBM干细胞,分别命名为SU5-1和SU5-2细胞。采用免疫荧光染色检测细胞巢蛋白、CD133的表达;诱导分化后采用免疫荧光染色检测细胞GFAP的表达,CCK-8实验检测细胞的生长曲线;Transwell侵袭实验检测细胞的侵袭能力;CCK-8实验检测细胞对替莫唑胺(TMZ)、卡铂(CBP)、顺铂(DDP)、阿霉素(ADM)的耐药性;Western blotting实验检测细胞程序性死亡受体-配体1(PD-L1)蛋白的表达;流式细胞仪检测细胞PD-L1阳性率的表达;基因检测分析同上。(3)建立裸小鼠原位初发及复发PDX模型,免疫组化染色检测PDX模型巢蛋白、GFAP、Ki-67的表达,基因检测分析同上。结果(1)与初发GBM比较,复发GBM标本Ki-67、巢蛋白阳性细胞数所占比例较高,而GFAP阳性细胞数所占比例较低,差异均有统计学意义(P<0.05);基因检测分析显示初发及复发GBM组织中IDH基因均无突变,初发GBM组织中MGMT基因呈甲基化状态,EGFR基因未扩增,而复发GBM组织中MGMT基因为非甲基化状态,EGFR基因扩增阳性。(2)SU5-1和SU5-2细胞均表达巢蛋白、CD133。诱导分化后均表达GFAP。生长曲线显示SU5-2细胞的增殖启动期早于SU5-1细胞,第3~5天两者持平,第6天起SU5-1细胞的增殖快于SU5-2细胞。SU5-2细胞的侵袭能力高于SU5-1细胞,差异有统计学意义(P<0.05)。5、10、15 mmol/L CBP、0.3125、1.25、5 mmol/L DDP,0.5、2 mmol/L ADM,125、500 mmol/L TMZ作用的SU5-2细胞的抑制率低于同浓
Objective To explore the molecular genetic characteristics of primary and recurrent glioblastomas (GBMs) from the same patient in vivo, primary glioma stem cells cultured in vitro, and patient-derived xenograft (PDX). Methods (1) The primary and recurrent GBM specimens from one patient during surgical resection were collected;and the expressions of glial fibrillary acidic protein (GFAP), nestin and Ki-67 were detected by immunohistochemical staining;the methylation of O6-methylguanine DNA methyltransferase (MGMT) gene, mutation of isocitrate dehydrogenase (IDH) gene and amplification of epidermal growth factor receptor (EGFR) gene were analyzed.(2) The primary and recurrent GBM stem cells were cultured in vitro and named as SU5-1 and SU5-2 cells, respectively;the expressions of nestin and CD133 were detected by immunohistochemical staining;GFAP expression was detected by immunohistochemical staining after induced differentiation, and the growth curve was detected by CCK-8 assay;Transwell invasion assay was used to detect the invasion ability;cell resistance to temozolomide (TMZ), carboplatin (CBP), cisplatin (DDP) and adriamycin (ADM) was detected by CCK-8 assay;the protein expression of programmed death receptor-ligand 1(PD-L1) was detected by Western blotting. The rate of PD-L1 positive cells was detected by flow cytometry;genetic testing analysis was as above.(3) The primary and recurrent in situ PDX models in nude mice were established, and the expressions of nestin, GFAP and Ki-67 were detected by immunohistochemical staining. Results (1) As compared with the primary GBM, the recurrent GBM had significantly higher percentages of Ki-67 and nestin positive cells, while statistically lower percentage of GFAP positive cells (P<0.05);genetic analysis showed that there was no mutation in IDH gene in the primary GBM tissues and recurrent GBM tissues;the MGMT gene in the primary GBM tissues was methylated and EGFR gene was not amplified, while the MGMT gene in recurrent GBM tissues was demethylated and EGFR gene amp
作者
施佳
董旭宸
戴晓晓
王海洋
代兴亮
刘加驰
姜倩倩
盛余敬
董军
Shi Jia;Dong Xuchen;Dai Xiaoxiao;Wang Haiyang;Dai Xingliang;Liu Jiachi;Jiang Qianqian;Sheng Yujing;Dong Jun(Department of Neurosurgery, Second Affiliated Hospital, Soochow University, Suzhou 215004, China;Department of Medicine, Soochow University, Suzhou 215000, China;Department of Pathology, Second Affiliated Hospital, Soochow University, Suzhou 215004, China)
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2019年第9期865-874,共10页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(81472739)
苏州市临床重点病种诊疗技术专项(LCZX201807).
