经鼻腔移植缺氧预处理的骨髓间充质干细胞对于小鼠脑出血作用的研究

发布时间：2018-04-14 17:41

本文选题：脑出血 + 缺氧预处理　；参考：《首都医科大学》2015年博士论文

【摘要】：研究背景脑出血占全部脑卒中的10%-15%，具有很高的死亡率及致残率，对患者、家庭及社会均可造成极大的负面影响和经济负担。目前，尚没有美国FDA批准的用于治疗脑出血的有效药物。近年来，干细胞移植已经成为治疗多种神经系统疾病的临床研究热点，用于脑出血的治疗亦逐渐受到研究者的关注。骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）是目前研究最多的一种干细胞。BMSCs具有向三个胚层分化的潜能，可自体取材移植，不需要组织配型、无免疫排斥等诸多问题，同时因其致癌性较低、来源丰富、培养条件相对简单等优势而成为研究热点。但是，对于治疗不同的疾病，如何选择适宜的BMSCs移植途径、剂量和时间，以及治疗的远期效果等问题还在不断的研究探索中。研究目的通过在体和离体实验，探讨经鼻腔途径移植缺氧预处理的骨髓间充质干细胞对小鼠脑出血的治疗作用及相关机制。实验方法实验共分为4个部分。 1．建立小鼠脑出血模型：用不同剂量的胶原酶IV立体定向注射至小鼠脑基底节区，采用病理解剖及行为学方法检测脑出血模型成功与否。 2．缺氧预处理BMSCs：取原代培养BMSCs，置入氧气浓度为0.5%的容器中24小时，应用MTT法检测缺氧预处理后的细胞活性，划痕试验检测细胞的迁移能力，Westernblot技术检测相关血管及神经生长因子的表达。 3．检测经鼻腔途径移植的HP-BMSCs：经鼻腔途径给予HP-BMSCs，荧光显微镜下沿嗅球、大脑皮层、血肿周围寻找荧光标记的HP-BMSCs，验证该方法的可靠性。 4．应用神经行为学、免疫组化及免疫印记等技术验证HP-BMSCs移植治疗效果：实验动物随机分为假手术组，对照组及治疗组。全部实验小鼠在建模前均进行行为学训练。（1）神经行为学检测方法包括mNss、Rotarod、贴纸去除实验以及旷场试验，分别检测不同时间点经鼻腔移植HP-BMSCs对于小鼠行为学的变化；（2）采用Western blot技术检测血肿灶周围神经生长因子，包括血管内皮生长因子（Vasclar Endothelial Growth Factor，VEGF）、脑源性神经生长因子（Brain-Derived Nerotrophic Factor，BDNF）和胶质细胞源性神经生长因子（Glial cell line-Derived Neurotrophic Factor，GDNF）的表达情况；（3）应用免疫组织化学技术检测侧脑室下区（Subventriclar Zone，SVZ）神经母细胞迁移和血肿灶周围新生神经元情况；（4）以Nissl染色技术评估小鼠脑出血后的病理改变。实验结果 1．以0.03U胶原酶IV立体定向注射至小鼠基底节区，脑出血模型稳定，神经功能缺损症状明显，死亡率低。 2．经缺氧预处理的BMSCs细胞活性及迁移能力明显增强，与对照组相比，BMSCs的促红细胞生成素（Erythropoietin，EPO），促红细胞生成素受体（Erythropoietin，receptor，EPOR）基质细胞衍生因子-1（SDF-1）表达明显上调。 3．在脑内嗅球、大脑皮层、基底节及血肿周围均可检测到经鼻腔移植HP-BMSCs，较多的HP-BMSCs聚集在血肿灶周围。 4．与对照组相比，HP-BMSCs治疗组小鼠神经功能缺损明显改善，SVZ区有更多的神经母细胞（BrdU+/DCX+共染细胞），，血肿周围出现更多的新生神经元（BrdU+/NeuN+共染细胞），血肿灶周围GDNF、VEGF及BDNF的表达明显上调，同时，ICH后21天HP-BMSCs治疗组小鼠基底节区体积缩小及侧脑室扩张程度明显减轻。结论缺氧预处理可显著提高BMSCs的细胞活力及生活学活性，经鼻腔移植的HP-BMSCs可以进入小鼠脑组织并迁移至血肿灶周围，改善ICH后神经功能缺损症状。HP-BMSCs可能通过上调相关神经生长因子的表达，促进内源性的神经再生进而加速神经功能的恢复。
[Abstract]:Research background
All stroke accounted for cerebral hemorrhage 10%-15%, high morbidity and mortality, is for patients, family and society can cause great negative impact and economic burden. At present, there is no effective drug approved by FDA for the treatment of cerebral hemorrhage. In recent years, stem cell transplantation has become a clinical research hotspot to cure many diseases the nervous system, for the treatment of cerebral hemorrhage has gradually drawn the attention of researchers.
Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) is a kind of stem cell research has the most.BMSCs to three differentiation ability, can be drawn without autologous transplantation, tissue typing, no immune rejection and many other issues, but because of its carcinogenic low, rich source, culture the condition is relatively simple and the advantage and become a research hotspot. However, for the treatment of different diseases, how to choose the proper ways of BMSCs transplantation, dose and time of continuous problems and the long-term effect of such treatment is still exploring.
research objective
In vivo and in vitro experiments were conducted to investigate the therapeutic effect and mechanism of bone marrow mesenchymal stem cells transplanting anoxic preconditioning through nasal passage on cerebral hemorrhage in mice.
Experimental method
The experiment is divided into 4 parts.
1., we established a mouse model of intracerebral hemorrhage: stereotactic injection of collagenase IV at different doses to the basal ganglia of mouse brain. We used pathological anatomy and behavioral methods to detect whether the cerebral hemorrhage model was successful or not.
2. hypoxia preconditioning BMSCs: Cultured in BMSCs, oxygen concentration of 0.5% containers in 24 hours, the cell activity of pretreatment was detected by MTT after hypoxia, the migration ability of cells was determined by scratch test, Westernblot detection of related blood vessels and nerve growth factor expression.
3. detection of HP-BMSCs through nasal cavity transplantation: HP-BMSCs was administered via nasal route, and fluorescent labeled HP-BMSCs was detected along the olfactory bulb, cerebral cortex and hematoma under fluorescence microscope, which verified the reliability of the method.
4. application of neuroethology, verify the effect of transplantation of HP-BMSCs immunohistochemistry and Western blot: the experimental animal were randomly divided into sham operation group, control group and treatment group. All mice in modeling were performed before behavioral training. (1) including mNss, detection method of neurobehavioral Rotarod removal experiment and stickers open field test were detected at different time points after transplantation for nasal HP-BMSCs changes the behavior of mouse; (2) detection in perihematoma region of nerve growth factor using Western blot technology, including vascular endothelial growth factor (Vasclar Endothelial, Growth Factor, VEGF), brain-derived neurotrophic factor (Brain-Derived Nerotrophic, Factor, BDNF) and glial cell derived neurotrophic factor (Glial cell line-Derived Neurotrophic Factor, GDNF) expression; (3) immunohistochemistry technique was used to detect the subventricular zone (Sub Ventriclar Zone, SVZ) migration of neuroblastoma and the status of newborn neurons around hematoma; (4) the pathological changes of cerebral hemorrhage in mice were evaluated by Nissl staining.
experimental result
1. with 0.03U collagenase IV stereotactic injection into the basal ganglia of mice, the model of cerebral hemorrhage was stable, the symptoms of nerve function defect were obvious, and the mortality rate was low.
2. the activity of BMSCs cells in hypoxia preconditioning and migration ability increased significantly, compared with the control group, BMSCs in epoetin (Erythropoietin, EPO), erythropoietin receptor (Erythropoietin, receptor, EPOR) stromal cell derived factor -1 (SDF-1) table Damien significantly up-regulated.
3. HP-BMSCs was detected around the olfactory bulb, cerebral cortex, basal ganglia and hematoma, and more HP-BMSCs was gathered around the hematoma.
4. compared with the control group, HP-BMSCs treatment group mice significantly improve neurologic impairment, SVZ more neuroblastoma (BrdU+/DCX+ staining cells), new neurons appear more around the hematoma (BrdU+/NeuN+, GDNF cells were stained) around hematoma lesions, significantly up-regulated the expression of VEGF and BDNF at the same time, 21 days after ICH HP-BMSCs treated mice in basal ganglia volume and dilated lateral ventricles were alleviated.
conclusion
Hypoxia preconditioning can significantly improve the cell viability and BMSCs activity of life, nasal HP-BMSCs transplantation can enter the brain and migrate to the surrounding hematoma lesions, improve the expression of ICH after neurological symptoms of.HP-BMSCs may be up-regulated related nerve growth factor, promote nerve regeneration of endogenous and accelerated the recovery of nerve function.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.34

【参考文献】