摘要
目的探索基于显微注射方法构建胶质母细胞瘤(GBM)融合大脑类器官模型的可行性,观察该模型肿瘤组织对替莫唑胺(TMZ)的敏感性。方法GBM组织和脑肿瘤旁组织标本(定义为正常脑组织)来源于中南大学湘雅医院神经外科行手术治疗的患者。采用绿色荧光蛋白(GFP)标记过的人诱导多能干细胞株(hiPSCs)培养24 d构建大脑类器官模型,通过免疫荧光染色方法检测蛋白标志物的表达,评估模型是否成功建立。采用显微注射方法将用红色荧光染料标记过的患者来源的GBM单细胞或U-251 MG细胞株注射到培养约30 d的大脑类器官中,构建GBM融合大脑类器官模型。采用免疫荧光染色法、HE染色等方法观察GBM细胞在大脑类器官中的增殖及浸润情况。采用免疫荧光染色法检测GBM融合大脑类器官中肿瘤细胞基质金属蛋白酶类(MMPs)及BrdU的表达情况,采用HE染色观察形态及结构变化。将10μmol/L TMZ加入GBM融合大脑类器官模型及GBM肿瘤类组织中培养48 h,采用免疫荧光染色法检测两种模型中Caspase-3的表达。结果(1)hiPSCs培养至第24天,形成具有明确芽和分层边缘的块状组织样结构;免疫荧光染色显示,该结构阳性表达前脑、脑室样结构、前额叶皮质及海马的标志蛋白FOXG1、N-cadherin、Auts2及Frizzled 9,以及星形胶质细胞标志蛋白胶质纤维酸性蛋白(GFAP)和神经元标志蛋白TUJ1。(2)荧光显微镜下及HE染色均显示肿瘤细胞在大脑类器官内快速增殖,呈浸润性生长。免疫荧光检测显示,与大脑类器官比较,GBM融合大脑类器官的肿瘤细胞中,MMP2和MMP13的表达明显增加,Fibronectin、MMP3及MMP9的表达明显减弱,差异均有统计学意义(均P<0.05),MMP10的表达差异无统计学意义(P>0.05);GFAP和BrdU的表达水平均增高(均P<0.05)。(3)TMZ处理48 h后,免疫荧光染色显示,GBM类肿瘤组织中Caspase-3的表达明显高于GBM融合大脑类器官。结论采用显微注射�
Objective To explore the feasibility of microinjection method to construct the brain organoids and glioblastoma co-culture system,and to evaluate its effect and sensitivity to temozolomide(TMZ)treatment.Methods GBM tissue samples and adjacent tissues were obtained from patients undergoing surgical treatment in the Neurosurgery Department of Xiangya Hospital,Central South University.The brain organoid model was constructed by human induced pluripotent cell line labeled with green fluorescent protein(GFP)after 24 days of culture.The expression of different protein markers was detected by immunofluorescence staining to evaluate whether the model was successfully established.We established a three-dimensional brain organoids and glioblastoma co-culture system by injecting tumor cells derived from patient-derived tumor cells or U-251 MG into cerebral organoids,in which tumor cells were labeled with red fluorescent dye.The proliferation and infiltration of GBM cells in cerebral organoids were observed by immunofluorescence staining and H-E staining.The expression of matrix metalloproteinases(MMPs),GFAP and BrdU of tumor cells in GBM fusion brain organ was detected by immunofluorescence staining,and the morphological and structural changes were observed by H-E staining.Tenμmol/L TMZ was added into the co-culture system and GBM organoids separately for 48 h,and the expression of Caspase-3 in the two models was detected by immunofluorescence staining.Results(1)After 24 days of organoid culture,a tissue-like structure with clear buds and stratified edges was formed.Immunofluorescence staining showed positive staining results for FOXG1,N-cadherin,Auts2,Frizzled 9,GFAP,and TUJ1,which were protein markers of forebrain,ventriculoid structure,prefrontal cortex,hippocampus,astrocyte,and neuron cells,respectively.(2)Transplanted GBM cells rapidly expanded within cerebral organoids and invaded into neural tissue.Compared with brain organoids,the expressions of MMP2 and MMP13 in GBM fused organoid tumor cells were significantly in
作者
黄兢
滕紫薇
张李洋
曾瑜
李成龙
刘志雄
刘方琨
Huang Jing;Teng Ziwei;Zhang Liyang;Zeng Yu;Li Chenglong;Liu Zhixiong;Liu Fangkun(Department of Neurosurgery,Xiangya Hospital,Central South University,Changsha 410008,China;Department of Psychiatry,the Second Xiangya Hospital,Central South University,Changsha 410011,China)
出处
《中华神经外科杂志》
CSCD
北大核心
2022年第5期492-499,共8页
Chinese Journal of Neurosurgery
基金
国家自然科学基金(81901401,82001223)。
