本文選題：缺血性腦卒中 + 大腦中動脈栓塞��； 參考：《上海交通大學》2014年博士論文

【摘要】：研究背景和目的 在中國，腦血管疾病(cerebral vascular diseases,CVD)在死亡原因中占據(jù)第三位，在血管性疾病致死的原因中占據(jù)首位。其中缺血性腦血管病(ischemic cerebralvaseular disease,ICD)占據(jù)所有腦血管病的70%左右，而在歐美國家則高達85%。目前治療ICD唯一有效并得到認可的的方法為發(fā)病急性期6小時內(nèi)進行溶栓治療，除此之外沒有其它有效的治療措施。最近十年干細胞移植治療缺血性腦卒中的實驗研究所取得成果及進展令人鼓舞。 骨髓單核細胞(bone marrow mononuclear cells，BMMNCs)取材容易、提取時間很短（1.5-3小時）、不需要培養(yǎng)、能夠自體抑制、沒有道德倫理學爭議。上述這些優(yōu)點使得BMMNCs成為干細胞移植戰(zhàn)略研究中非常有潛力的移植細胞，也使之成為移植治療缺血性腦卒中較佳的種子細胞。BMMNCs移植治療缺血性腦卒中的機制尚不清楚,目前更多的研究結(jié)果支持其是通過分泌一系列營養(yǎng)因子而促進神經(jīng)修復�；谶@個理論假設(shè)，具有較強分泌營養(yǎng)因子能力的BMMNCs可以認為是目前最佳的治療缺血性腦卒中的種子細胞。 我們在前期研究中首次發(fā)現(xiàn)與普通的BMMNCs相比，骨髓抑制-再生的單核細胞（bone marrow regenerative mononuclear cells，BMRMNCs）具有更強的分泌營養(yǎng)因子的能力（如bFGF、NGF、BDNF、VEGF等），故我們推測其移植后能夠遷移到缺血區(qū)并通過分泌營養(yǎng)因子促進血管再生、抑制細胞凋亡和炎性反應而促進神經(jīng)功能恢復。為證實這一推測，本研究建立局灶性腦缺血模型并移植BMRMNCs，從細胞-功能-整體三個層次，移植前后分別觀測BMRMNCs在體外和模型缺血區(qū)分泌營養(yǎng)因子的情況，及探索這些營養(yǎng)因子對缺血區(qū)細胞凋亡、炎癥反應、血管再生的影響，從而證實BMRMNCs與一般的BMMNCs相比具有更強的分泌營養(yǎng)因子的能力，為臨床治療缺血性腦卒中提供最佳種子細胞。 研究內(nèi)容和方法 通過建立大鼠骨髓抑制-再生模型獲得BMRMNCs；通過改良大鼠大腦中動脈栓塞（middle cerebral artery occlusion，MCAO）模型制作方法獲得更加穩(wěn)定的局灶性腦缺血再灌注模型；經(jīng)靜脈移植BMRMNCs入局灶性腦缺血再灌注模型大鼠后觀察BMRMNCs的療效。 課題研究共分為五部分:第一部分：通過一次性尾靜脈注射5-氟脲嘧啶（150mg/kg）建立大鼠骨髓抑制-再生模型并動態(tài)觀察骨髓變化情況。第二部分：對傳統(tǒng)MCAO模型制作方法進行改良，以獲得更穩(wěn)定更可靠的局灶性腦缺血再灌注損傷模型。第三部分：通過對BMRMNCs體外營養(yǎng)因子分泌情況及細胞成分的檢測以了解BMRMNCs生物學功能。第四部分：通過對靜脈移植后BMRMNCs的跟蹤了解其能否遷移到缺血區(qū)以及其趨化、遷移的機制。第五部分：BMRMNCs經(jīng)靜脈移植入MCAO大鼠后，檢測其在模型缺血區(qū)分泌營養(yǎng)因子的情況，并觀察這些營養(yǎng)因子對缺血區(qū)細胞凋亡、炎癥反應、血管再生的影響以進一步探索BMRMNCs治療缺血性腦卒中的機制。 研究結(jié)果 1．5-Fu注射后大鼠外周血白細胞、血小板計數(shù)及骨髓細胞計數(shù)均急劇下降,于第7d下降到最低點后逐步恢復并于第14d恢復到正常水平，上述改變符合經(jīng)典的骨髓抑制-再生過程，提示骨髓抑制-再生模型制作成功。 2．對大鼠大腦中動脈栓塞（middle cerebral artery occlusion，MCAO）模型制作方法進行改良，顯著提高了模型腦梗死體積和神經(jīng)功能學評分的穩(wěn)定性，，增加了實驗數(shù)據(jù)的可靠性，為后續(xù)實驗提供一個標準化的平臺。 3．BMRMNCs體外分泌VEGF、bFGF、BDNF、NGF的水平明顯提高，而且其細胞中CD34+、CD45+、CD90+細胞含量增多，提示其各種干細胞的含量增多。 4．靜脈移植后與BMMNCs相比，有更多的BMRMNCs遷移到缺血區(qū)并存活。BMRMNCs遷移能力的增強可能與其表達的趨化因子SDF-1的受體CXCR4增多相關(guān)。 5．與BMMNCs相比，BMRMNCs對MCAO大鼠有更好的治療效果。BMRMNCs移植后通過檢測發(fā)現(xiàn)：缺血區(qū)分泌的營養(yǎng)因子及微血管數(shù)目明顯增多，而凋亡細胞和炎性因子明顯減少，移植的細胞只有很小的比率轉(zhuǎn)分化為神經(jīng)細胞及神經(jīng)膠質(zhì)細胞。這提示BMRMNCs不是通過細胞替代而是通過分泌營養(yǎng)因子進一步抑制缺血區(qū)細胞凋亡和炎性反應并促進血管再生而發(fā)揮治療作用。 結(jié)論 與BMMNCs相比，BMRMNCs在體外具有更強的營養(yǎng)因子分泌能力和趨化遷移到病灶的能力。經(jīng)靜脈移植后BMRMNCs能遷移到宿主腦內(nèi)缺血區(qū)并分泌較高水平的營養(yǎng)因子，能減少半暗帶區(qū)細胞凋亡、促進半暗帶區(qū)血管再生、減少半暗帶區(qū)炎癥反應、減少腦梗死體積并促進行為學的恢復，其治療效果優(yōu)于普通的BMMNCs。BMRMNCs可作為臨床治療缺血性腦卒中較佳的種子細胞。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743.3

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