原代培养细胞建立脑干胶质瘤动物模型及在放、化疗联合治疗中的初步应用

Brainstem gliomas is among the most malignant neoplasms in the central nervous system with pretty poor prognosis.Recent researches revealed that their inherent biology were quiet different from the supratentorial gliomas.Brainstem glioma animal models were induced by sepecific gene knocking-out or injetion glioma cell lines or primary culture cells from supratentorial gliomas according to present publications.Those models could fit their experimental objectives,while could not reflect their inherent invasiveness characters completely or evaluate radiotherapy or chemotherapy subjectively.Stereotactic injection of human brainstem glioma primary cultured cells into nude mice brainstem to construct animal models have not been published yet, according to our literature review.This program is going to improve fresh glioma tissue isolation techniques based on our previous research works and to adapt cell culture environments so that setting up stable human brainstem glioma cell lines which would be tested in the following experiments;to set up nude mice brainstem glioma model by stereotactic injection human brainstem glioma cells which would be highly consistent with its clinical origins and then be confirmed by animal Basso-Beattie-Bresnahan scale,MRI,histology and gene assay,so that a construction protocol will be formed finally;the minimal brainstem glioma tissue could also be expanded by the tumor growth in mice model,in this way,more tissue could be achieved for further researches;the animal model will be divided into several groups to underwent radiotherpay,chenmotherapy or radio-chemotherapy respectively, the treatment results will be compared to build the most effective treatment strategies for this kind of malignant tumours in braistem. All these researches will establish a good basement for future researches on improving individual treatment strategies for patients.

脑干胶质瘤是中枢神经系统治疗效果最差的恶性肿瘤之一，目前研究认为其具有不同于大脑半球胶质瘤的独特生物学特性。现有文献报道所用的基因诱导或采用商品化细胞系及大脑半球胶质瘤来源的细胞建立动物模型，未能保留脑干胶质瘤固有生物学特性，不能客观评价治疗效果。采用脑干胶质瘤原代培养细胞制备原位动物模型，国内外尚未见报道。本项目拟在前期研究的基础上，改进人脑干胶质瘤新鲜组织分离技术，调整培养条件，建立稳定的脑干胶质瘤原代培养细胞系，并检测其生物学特性，立体定向注射到裸鼠脑干，建立保留其固有生物学特性及临床特征的脑干胶质瘤原位动物模型，采用动物行为、MRI、组织形态学及基因检测等方法进行评估比较，建立规范化生产流程；通过动物模型实现脑干微小肿瘤组织的体外扩增，克服脑干胶质瘤标本微小对后续研究的限制；对荷瘤动物随机分组，分别进行长期生存观察、放射、化学治疗及放化疗结合等治疗策略效果比较，为个体化治疗奠定基础

本项目历时1年，目前完成了课题部分内容，其中脑干胶质瘤原代培养是本课题难点，目前可以传代4-5代，传代周期为7-9天，但仍需进一步优化培养条件；同时，我们应用U87胶质瘤细胞系成功建立了稳定可复制的裸大鼠脑干胶质瘤动物模型，并进行侵袭性差异比较研究，为下一步建立原代培养细胞动物模型建立了基础。另一方面，我们通过对脑干胶质瘤干细胞的分离、培养及鉴定，初步证实脑干胶质瘤中存在一类比例较少、具有自我更新和增殖潜能并表达Nestin和CD133，具有干细胞特征的肿瘤细胞，这种肿瘤干细胞具有多向分化能力，分化的产物是不成熟的肿瘤前体细胞，其具有亲本肿瘤的特性，在体外可将其分离培养传代。耐药试验表明，脑干胶质瘤干细胞对替莫唑胺有较强的抗药性；同时有较强的抗营养缺乏能力，这可能是脑干脑干胶质瘤对替莫唑胺化疗不敏感的原因之一。