骨髓抑制—再生单核细胞移植治疗局灶性脑缺血再灌注损伤模型的研究

发布时间：2018-07-05 09:55

本文选题：缺血性脑卒中 + 大脑中动脉栓塞　；参考：《上海交通大学》2014年博士论文

【摘要】：研究背景和目的在中国，脑血管疾病(cerebral vascular diseases,CVD)在死亡原因中占据第三位，在血管性疾病致死的原因中占据首位。其中缺血性脑血管病(ischemic cerebralvaseular disease,ICD)占据所有脑血管病的70%左右，而在欧美国家则高达85%。目前治疗ICD唯一有效并得到认可的的方法为发病急性期6小时内进行溶栓治疗，除此之外没有其它有效的治疗措施。最近十年干细胞移植治疗缺血性脑卒中的实验研究所取得成果及进展令人鼓舞。骨髓单核细胞(bone marrow mononuclear cells，BMMNCs)取材容易、提取时间很短（1.5-3小时）、不需要培养、能够自体抑制、没有道德伦理学争议。上述这些优点使得BMMNCs成为干细胞移植战略研究中非常有潜力的移植细胞，也使之成为移植治疗缺血性脑卒中较佳的种子细胞。BMMNCs移植治疗缺血性脑卒中的机制尚不清楚,目前更多的研究结果支持其是通过分泌一系列营养因子而促进神经修复。基于这个理论假设，具有较强分泌营养因子能力的BMMNCs可以认为是目前最佳的治疗缺血性脑卒中的种子细胞。我们在前期研究中首次发现与普通的BMMNCs相比，骨髓抑制-再生的单核细胞（bone marrow regenerative mononuclear cells，BMRMNCs）具有更强的分泌营养因子的能力（如bFGF、NGF、BDNF、VEGF等），故我们推测其移植后能够迁移到缺血区并通过分泌营养因子促进血管再生、抑制细胞凋亡和炎性反应而促进神经功能恢复。为证实这一推测，本研究建立局灶性脑缺血模型并移植BMRMNCs，从细胞-功能-整体三个层次，移植前后分别观测BMRMNCs在体外和模型缺血区分泌营养因子的情况，及探索这些营养因子对缺血区细胞凋亡、炎症反应、血管再生的影响，从而证实BMRMNCs与一般的BMMNCs相比具有更强的分泌营养因子的能力，为临床治疗缺血性脑卒中提供最佳种子细胞。研究内容和方法通过建立大鼠骨髓抑制-再生模型获得BMRMNCs；通过改良大鼠大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型制作方法获得更加稳定的局灶性脑缺血再灌注模型；经静脉移植BMRMNCs入局灶性脑缺血再灌注模型大鼠后观察BMRMNCs的疗效。课题研究共分为五部分:第一部分：通过一次性尾静脉注射5-氟脲嘧啶（150mg/kg）建立大鼠骨髓抑制-再生模型并动态观察骨髓变化情况。第二部分：对传统MCAO模型制作方法进行改良，以获得更稳定更可靠的局灶性脑缺血再灌注损伤模型。第三部分：通过对BMRMNCs体外营养因子分泌情况及细胞成分的检测以了解BMRMNCs生物学功能。第四部分：通过对静脉移植后BMRMNCs的跟踪了解其能否迁移到缺血区以及其趋化、迁移的机制。第五部分：BMRMNCs经静脉移植入MCAO大鼠后，检测其在模型缺血区分泌营养因子的情况，并观察这些营养因子对缺血区细胞凋亡、炎症反应、血管再生的影响以进一步探索BMRMNCs治疗缺血性脑卒中的机制。研究结果 1．5-Fu注射后大鼠外周血白细胞、血小板计数及骨髓细胞计数均急剧下降,于第7d下降到最低点后逐步恢复并于第14d恢复到正常水平，上述改变符合经典的骨髓抑制-再生过程，提示骨髓抑制-再生模型制作成功。 2．对大鼠大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型制作方法进行改良，显著提高了模型脑梗死体积和神经功能学评分的稳定性，，增加了实验数据的可靠性，为后续实验提供一个标准化的平台。 3．BMRMNCs体外分泌VEGF、bFGF、BDNF、NGF的水平明显提高，而且其细胞中CD34+、CD45+、CD90+细胞含量增多，提示其各种干细胞的含量增多。 4．静脉移植后与BMMNCs相比，有更多的BMRMNCs迁移到缺血区并存活。BMRMNCs迁移能力的增强可能与其表达的趋化因子SDF-1的受体CXCR4增多相关。 5．与BMMNCs相比，BMRMNCs对MCAO大鼠有更好的治疗效果。BMRMNCs移植后通过检测发现：缺血区分泌的营养因子及微血管数目明显增多，而凋亡细胞和炎性因子明显减少，移植的细胞只有很小的比率转分化为神经细胞及神经胶质细胞。这提示BMRMNCs不是通过细胞替代而是通过分泌营养因子进一步抑制缺血区细胞凋亡和炎性反应并促进血管再生而发挥治疗作用。结论与BMMNCs相比，BMRMNCs在体外具有更强的营养因子分泌能力和趋化迁移到病灶的能力。经静脉移植后BMRMNCs能迁移到宿主脑内缺血区并分泌较高水平的营养因子，能减少半暗带区细胞凋亡、促进半暗带区血管再生、减少半暗带区炎症反应、减少脑梗死体积并促进行为学的恢复，其治疗效果优于普通的BMMNCs。BMRMNCs可作为临床治疗缺血性脑卒中较佳的种子细胞。
[Abstract]:Background and purpose of research
In China, cerebral vascular diseases (CVD) occupies third of the causes of death, and occupies the first place in the cause of death of vascular diseases. Ischemic cerebrovascular disease (ischemic cerebralvaseular disease, ICD) occupies 70% left right of all cerebrovascular diseases, while in European and American countries, the only present treatment of ICD is that of ICD. The effective and recognised method of thrombolytic therapy within 6 hours of the acute phase of the onset is that there are no other effective treatments. The results and progress of the last ten years of stem cell transplantation for the treatment of ischemic stroke are encouraging.
Bone marrow mononuclear cells (bone marrow mononuclear cells, BMMNCs) are easy to be obtained, the extraction time is very short (1.5-3 hours), it does not need to be cultured, it can be self suppressed, and there is no moral ethical dispute. These advantages make BMMNCs a very potential transplant cell in the study of stem cell transplantation strategy, and make it a transplantation therapy for ischemia. The mechanism of cerebral apoplexy with better seed cell.BMMNCs transplantation is not clear, and more research results support it to promote nerve repair by secreting a series of nutrient factors. Based on this theory, BMMNCs with strong secretory factor ability can be considered as the best treatment of ischemic disease. A seed cell of a stroke.
In our previous study, we first found that bone marrow suppression - regenerated mononuclear cells (bone marrow regenerative mononuclear cells, BMRMNCs) have a stronger ability to secrete nutritional factors (such as bFGF, NGF, BDNF, VEGF, etc.) compared with common BMMNCs, so we speculate that after transplantation it can migrate into the ischemic area and secrete nutritional factors. In order to confirm this hypothesis, this study established the focal cerebral ischemia model and transplanted BMRMNCs, from the three levels of cell function to the whole, to observe the secretion of nutrient factors by BMRMNCs in vitro and model ischemic areas, and to explore these nutrients. The effect of factors on apoptosis, inflammatory response, and vascular regeneration in ischemic area confirms that BMRMNCs has a stronger ability to secrete nutritional factors than general BMMNCs, and provides the best seed cells for clinical treatment of ischemic stroke.
Research contents and methods
BMRMNCs was obtained by establishing a rat bone marrow suppression and regeneration model, and a more stable focal cerebral ischemia reperfusion model was obtained by improving the middle cerebral artery occlusion (MCAO) model of the rat middle cerebral artery (MCAO) model. After intravenous BMRMNCs into the focal cerebral ischemia reperfusion model in rats, the treatment of BMRMNCs therapy was observed. Effect.
The research is divided into five parts: the first part: the rat bone marrow suppression regeneration model was established by injection of 5- fluorouracil (150mg/kg) in the tail vein and the change of bone marrow was observed dynamically. The second part: to improve the traditional MCAO model making method to obtain a more stable and more reliable model of focal cerebral ischemia reperfusion injury The third part: to understand the biological function of BMRMNCs by the detection of the secretion of BMRMNCs extracorporeal nutrition factor and the detection of cell components. Fourth: through the tracking of BMRMNCs after the vein transplantation to understand whether it can migrate into the ischemic area and its chemotaxis and migration mechanism. Part fifth: after the BMRMNCs transplants into the MCAO rats, the test is detected. The effect of these nutrients on apoptosis, inflammatory response, and vascular regeneration in ischemic region was observed to further explore the mechanism of BMRMNCs in the treatment of ischemic stroke.
Research results
After 1.5-Fu injection, the peripheral blood leucocyte, platelet count and bone marrow cell count declined sharply. After the descent of 7D to the lowest point, it recovered gradually and recovered to the normal level in 14d. The changes conformed to the classic bone marrow suppression regeneration process, suggesting the success of the bone marrow suppression regeneration model.
2. the improvement of the middle cerebral artery occlusion (MCAO) model of rat model of cerebral artery embolism significantly improved the stability of the volume of cerebral infarction and the score of neurologic function, increased the reliability of the experimental data, and provided a standardized platform for the follow-up experiment.
The level of VEGF, bFGF, BDNF and NGF increased in 3.BMRMNCs in vitro, and the content of CD34+, CD45+ and CD90+ cells in the cells increased, suggesting that the content of all kinds of stem cells increased.
4. after 4. intravenous transplantation, more BMRMNCs migrated to the ischemic zone and increased the ability to survive.BMRMNCs migration may be associated with the increase of the receptor CXCR4 of the chemokine SDF-1.
5. compared with BMMNCs, BMRMNCs had better therapeutic effect on MCAO rats. After.BMRMNCs transplantation, it was found that the number of nutrient factors and the number of microvessels in the ischemic region increased significantly, while the apoptotic cells and inflammatory factors decreased significantly, and the transplanted cells had only a very small ratio to differentiate into nerve cells and glial cells. This suggested that BM RMNCs is not a cell replacement but a therapeutic effect by secreting nutrient factors to further inhibit apoptosis and inflammatory responses in ischemic areas and promote vascular regeneration.
conclusion
Compared with BMMNCs, BMRMNCs has a stronger ability of nutrient secretion and chemotaxis to migrate to the focus in vitro. After transplanting, BMRMNCs can migrate to the host brain ischemia area and secrete a high level of nutrient factors. It can reduce cell apoptosis in the semi dark zone, promote the regeneration of the blood vessels in the semi dark zone, reduce the inflammatory response in the semi dark zone, and reduce the inflammatory response in the darker zone. The volume of less cerebral infarction and the promotion of the recovery of behavior are better than the common BMMNCs.BMRMNCs, which can be used as a better seed cell for the treatment of ischemic stroke.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】