BDNF转基因型温敏干细胞在脑创伤半损伤带低温环境下的移植分化研究

项目名称： BDNF转基因型温敏干细胞在脑创伤半损伤带低温环境下的移植分化研究

项目编号： No.30872668

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 张赛

作者单位： 中国人民武装警察部队后勤学院

项目金额： 35万元

中文摘要： 目的：建立一种温度敏感型干细胞系，同时利用脑源性神经营养因子（BDNF）的营养和诱导作用，增加移植干细胞向神经元方向的分化比例和促进创伤性脑损伤（TBI）的恢复。 方法：分离和鉴定人源性脐带间充质干细胞（UCMSCs）。tsSV40LT质粒转染UCMSCs（ts-UCSMCs）；检测ts-UCSMCs的生长特性；利用腺病毒载体转染BDNF进入ts-UCSMCs；建立去胸腺小鼠颅脑液压打击模型进行干细胞原位移植，免疫组化检测脑组织标本PCNA及神经细胞分化指标GFAP、NSE和nestin的表达水平，实验动物予以神经功能缺陷评分。 结果：成功获得UCMSCs和ts-UCSMCs，且ts-UCSMCs的生长呈温度依赖性。动物移植结果显示，与UCSMCs相比，ts-UCSMCs组在33℃#26102;PCNA表达明显增高，而BDNF-ts-UCSMCs进一步提高神经元标志NSE的比例，并降低神经功能缺陷评分。 结论：ts-UCSMCs移植联合TBI后亚低温治疗可提高UCSMCs在体内的存活和分化，而BDNF的应用可进一步提高干细胞向神经元分化的比例和促进TBI后神经功能的恢复。

中文关键词： 创伤性脑损伤；干细胞；移植；低温疗法；脑源性神经生长因子

英文摘要： Objective：To study the proliferation and differentiation character of a temperature-sensitive stem cell line cultured at 33℃and 37℃respectively for establishing a basement on stem cells transplantation into injured brain area during hypothermia treatment. The brain derived neurotrophic factor (BDNF) was transferred into human umbilical cord mesenchymal stem cells (UCMSCs) for promoting the stem cells differentiation into neuron and enhance neuromotor function after head injury. Methods: UCMSCs were isolated and expanded in culture, and certificated by flow cytometry analysis. After transfecting plasmid containing temperature-sensitive simian virus 40 large T-antigen (tsSV40LT) into UCMSCs (ts-UCMSCs), growth characterization of ts-UCMSCs was analyzed. Recombinant adenovirus-mediated BDNF gene transferred into ts-UCMSCs. After TBI models in athymic mice were established, UCMSCs, ts-UCMSCs or BDNF-ts-UCMSCs were transplanted into injured area. Proliferating cell nuclear antigen (PCNA), nestin, GFAP and NSE expression were detected by immunofluorescent staining. Results: The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at 33℃incubator, ts-UCMSCs displayed high proliferation activity, strong tolerance to low nutrient conditions, and strong cell clone ability, as well as its telomerase activation, highly expressed cyclin D1 and CDK2, and lowly expressed P16, meanwhile, highly nestin, lowly NSE and GFAP. But when cultured at 37℃incubator, the cell line showed a completely converse profile. The BDNF was transfected successfully into ts-UCMSCs. Compared with UCSMCs group, the level of PCNA in injured region transplanted with ts-UCSMCs elevated obviously with mild hypothermia therapy. However, transplantation of BDNF-ts-UCMSCs could further increase the expression of neural marker NSE and decrease the score of neurological impairment compared with ts-UCMSCs group. Conclusion: Combination of ts-UCSMCs and mild hypothermia therapy after TBI could facilitate the survival rate and differentiation of UCSMCs. Addition of BDNF could further promote the differentiation of neurons of ts-UCSMCs and recovery of neurological function after TBI.

英文关键词： Traumatic Brain Injury; Stem Cell; Transplantation; Hypothermia Therapy; BDNF