本文選題：腦出血 + 缺氧預處理�。� 參考：《首都醫(yī)科大學》2015年博士論文

【摘要】：研究背景 腦出血占全部腦卒中的10%-15%，具有很高的死亡率及致殘率，對患者、家庭及社會均可造成極大的負面影響和經(jīng)濟負擔。目前，尚沒有美國FDA批準的用于治療腦出血的有效藥物。近年來，干細胞移植已經(jīng)成為治療多種神經(jīng)系統(tǒng)疾病的臨床研究熱點，用于腦出血的治療亦逐漸受到研究者的關(guān)注。 骨髓間充質(zhì)干細胞（Bone marrow mesenchymal stem cells，BMSCs）是目前研究最多的一種干細胞。BMSCs具有向三個胚層分化的潛能，可自體取材移植，不需要組織配型、無免疫排斥等諸多問題，同時因其致癌性較低、來源豐富、培養(yǎng)條件相對簡單等優(yōu)勢而成為研究熱點。但是，對于治療不同的疾病，如何選擇適宜的BMSCs移植途徑、劑量和時間，以及治療的遠期效果等問題還在不斷的研究探索中。 研究目的 通過在體和離體實驗，探討經(jīng)鼻腔途徑移植缺氧預處理的骨髓間充質(zhì)干細胞對小鼠腦出血的治療作用及相關(guān)機制。 實驗方法 實驗共分為4個部分。 1．建立小鼠腦出血模型：用不同劑量的膠原酶IV立體定向注射至小鼠腦基底節(jié)區(qū)，采用病理解剖及行為學方法檢測腦出血模型成功與否。 2．缺氧預處理BMSCs：取原代培養(yǎng)BMSCs，置入氧氣濃度為0.5%的容器中24小時，應用MTT法檢測缺氧預處理后的細胞活性，劃痕試驗檢測細胞的遷移能力，Westernblot技術(shù)檢測相關(guān)血管及神經(jīng)生長因子的表達。 3．檢測經(jīng)鼻腔途徑移植的HP-BMSCs：經(jīng)鼻腔途徑給予HP-BMSCs，熒光顯微鏡下沿嗅球、大腦皮層、血腫周圍尋找熒光標記的HP-BMSCs，驗證該方法的可靠性。 4．應用神經(jīng)行為學、免疫組化及免疫印記等技術(shù)驗證HP-BMSCs移植治療效果：實驗動物隨機分為假手術(shù)組，對照組及治療組。全部實驗小鼠在建模前均進行行為學訓練。（1）神經(jīng)行為學檢測方法包括mNss、Rotarod、貼紙去除實驗以及曠場試驗，分別檢測不同時間點經(jīng)鼻腔移植HP-BMSCs對于小鼠行為學的變化；（2）采用Western blot技術(shù)檢測血腫灶周圍神經(jīng)生長因子，包括血管內(nèi)皮生長因子（Vasclar Endothelial Growth Factor，VEGF）、腦源性神經(jīng)生長因子（Brain-Derived Nerotrophic Factor，BDNF）和膠質(zhì)細胞源性神經(jīng)生長因子（Glial cell line-Derived Neurotrophic Factor，GDNF）的表達情況；（3）應用免疫組織化學技術(shù)檢測側(cè)腦室下區(qū)（Subventriclar Zone，SVZ）神經(jīng)母細胞遷移和血腫灶周圍新生神經(jīng)元情況；（4）以Nissl染色技術(shù)評估小鼠腦出血后的病理改變。 實驗結(jié)果 1．以0.03U膠原酶IV立體定向注射至小鼠基底節(jié)區(qū)，腦出血模型穩(wěn)定，神經(jīng)功能缺損癥狀明顯，死亡率低。 2．經(jīng)缺氧預處理的BMSCs細胞活性及遷移能力明顯增強，與對照組相比，BMSCs的促紅細胞生成素（Erythropoietin，EPO），促紅細胞生成素受體（Erythropoietin，receptor，EPOR）基質(zhì)細胞衍生因子-1（SDF-1）表達明顯上調(diào)。 3．在腦內(nèi)嗅球、大腦皮層、基底節(jié)及血腫周圍均可檢測到經(jīng)鼻腔移植HP-BMSCs，較多的HP-BMSCs聚集在血腫灶周圍。 4．與對照組相比，HP-BMSCs治療組小鼠神經(jīng)功能缺損明顯改善，SVZ區(qū)有更多的神經(jīng)母細胞（BrdU+/DCX+共染細胞），，血腫周圍出現(xiàn)更多的新生神經(jīng)元（BrdU+/NeuN+共染細胞），血腫灶周圍GDNF、VEGF及BDNF的表達明顯上調(diào)，同時，ICH后21天HP-BMSCs治療組小鼠基底節(jié)區(qū)體積縮小及側(cè)腦室擴張程度明顯減輕。 結(jié)論 缺氧預處理可顯著提高BMSCs的細胞活力及生活學活性，經(jīng)鼻腔移植的HP-BMSCs可以進入小鼠腦組織并遷移至血腫灶周圍，改善ICH后神經(jīng)功能缺損癥狀。HP-BMSCs可能通過上調(diào)相關(guān)神經(jīng)生長因子的表達，促進內(nèi)源性的神經(jīng)再生進而加速神經(jīng)功能的恢復。
[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.

【學位授予單位】：首都醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R743.34

【參考文獻】

相關(guān)期刊論文 前5條

1 張芹;張擁波;李繼梅;;腦出血研究進展[J];神經(jīng)損傷與功能重建;2013年06期

2 曹文鋒;謝旭芳;張洪連;張昆南;吳曉牧;;自體骨髓間充質(zhì)干細胞移植治療腦出血臨床研究[J];中國實用神經(jīng)疾病雜志;2014年07期

3 閆峰;吉訓明;羅玉敏;;多種實驗動物腦出血模型的制作[J];實驗動物科學;2009年01期

4 張平;張本恕;;間充質(zhì)干細胞的特性及臨床應用前景[J];中國老年學雜志;2013年03期

5 王利平;趙景敏;樸哲;南光賢;;自體骨髓間充質(zhì)干細胞移植治療腦卒中的療效[J];中國老年學雜志;2013年16期

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