本文選題：創(chuàng)傷性腦損傷 + 內(nèi)皮祖細(xì)胞�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:顱腦創(chuàng)傷(Traumaticbraininjury,TBI)嚴(yán)重危害人類健康,具有較高的致殘率和死亡率。顱腦創(chuàng)傷后神經(jīng)血管的修復(fù)決定患者的預(yù)后,其中創(chuàng)傷灶周?chē)艿男迯?fù)又是其關(guān)鍵影響因素之一。本課題組前期研究證實(shí)顱腦創(chuàng)傷后內(nèi)皮祖細(xì)胞(endothelialprogenitor cells,EPCs)動(dòng)員增加,且在外周血中呈現(xiàn)先抑后揚(yáng)的變化趨勢(shì),也有研究證實(shí)內(nèi)皮祖細(xì)胞能夠歸巢定位到創(chuàng)傷灶局部,促進(jìn)創(chuàng)傷灶周?chē)难苄律?但是內(nèi)皮祖細(xì)胞是否直接分化為血管內(nèi)皮細(xì)胞并參與血管新生的機(jī)制目前仍不清楚。已有的報(bào)道證明了內(nèi)皮祖細(xì)胞是通過(guò)旁分泌作用促進(jìn)損傷區(qū)血管殘端的內(nèi)皮細(xì)胞增殖分化形成新生的微血管,而不是直接分化為血管內(nèi)皮細(xì)胞參與修復(fù)作用。內(nèi)皮祖細(xì)胞被證實(shí)可以分泌外泌體,并且內(nèi)皮祖細(xì)胞外泌體通過(guò)其內(nèi)包含的多種生物學(xué)物質(zhì)發(fā)揮作用,但是內(nèi)皮祖細(xì)胞來(lái)源的外泌體在促進(jìn)腦創(chuàng)傷后的神經(jīng)血管修復(fù)中的作用機(jī)制尚不明確。本實(shí)驗(yàn)通過(guò)觀察內(nèi)皮祖細(xì)胞外泌體在體內(nèi)外實(shí)驗(yàn)觀察內(nèi)皮祖細(xì)胞外泌體對(duì)損傷內(nèi)皮細(xì)胞的作用及其機(jī)制,為應(yīng)用內(nèi)皮祖細(xì)胞外泌體治療創(chuàng)傷性腦損傷提供理論參考。方法:(1)應(yīng)用梯度密度離心的方法體外獲取臍帶血單個(gè)核細(xì)胞(MNCs),培養(yǎng)純化內(nèi)皮祖細(xì)胞并鑒定其功能;(2)經(jīng)超速離心的方法獲取體外培養(yǎng)的內(nèi)皮祖細(xì)胞分泌到培養(yǎng)基中的外泌體,應(yīng)用透射電鏡、westernblot的方法觀察檢測(cè)內(nèi)皮祖細(xì)胞外泌體形態(tài)特征以及表面特異蛋白分子。(3)借助低氧培養(yǎng)箱體外制作的內(nèi)皮細(xì)胞低氧損傷模型,應(yīng)用Western blot的方法檢測(cè)內(nèi)皮祖細(xì)胞外泌體干預(yù)后其緊密連接蛋白的表達(dá)變化以及促進(jìn)血管生成相關(guān)蛋白的表達(dá)情況。(4)應(yīng)用腦皮質(zhì)損傷儀(CCI)制備小鼠顱腦損傷模型,穿刺小鼠尾靜脈移植內(nèi)皮祖細(xì)胞外泌體,應(yīng)用免疫熒光和westernblot的方法檢測(cè)移植內(nèi)皮祖細(xì)胞外泌體后的腦創(chuàng)傷小鼠創(chuàng)傷灶周?chē)律苊芏?緊密連接蛋白的變化,血腦屏障伊文思藍(lán)的滲漏,應(yīng)用PCR技術(shù)檢測(cè)緊密連接蛋白、MMP9、VEGF表達(dá)情況,應(yīng)用Western blot技術(shù)檢測(cè)促血管生成蛋白PTEN表達(dá)水平變化。結(jié)果:成功的應(yīng)用梯度密度離心的方法體外獲取臍帶血單個(gè)核細(xì)胞,并在體外擴(kuò)增培養(yǎng);經(jīng)超速離心的方法獲取內(nèi)皮祖細(xì)胞來(lái)源的外泌體,通過(guò)透射電鏡、westernblot的方法鑒定內(nèi)皮祖細(xì)胞能夠分泌外泌體;利用熒光染料PKH26對(duì)外泌體進(jìn)行標(biāo)記,證實(shí)內(nèi)皮細(xì)胞能夠吞噬內(nèi)皮祖細(xì)胞外泌體;體外實(shí)驗(yàn)證實(shí)內(nèi)皮祖細(xì)胞促進(jìn)損傷內(nèi)皮細(xì)胞緊密連接蛋白CLN5、ZO-1、OCLN表達(dá)增高,PTEN表達(dá)水平降低,p-Akt水平升高。體內(nèi)實(shí)驗(yàn)提示臍帶血內(nèi)皮祖細(xì)胞外泌體能夠減輕進(jìn)腦創(chuàng)傷后血腦屏障伊文思藍(lán)滲透率;PCR技術(shù)證實(shí)創(chuàng)傷灶緊密連接蛋白CLN5、ZO-1、OCLN表達(dá)增高,VEGF表達(dá)水平增高,MMP9表達(dá)水平升高;Western blot表明外泌體能夠減少第3天和第7天創(chuàng)傷灶PTEN的表達(dá)水平。結(jié)論:內(nèi)皮祖細(xì)胞能夠分泌大量的外泌體,體外實(shí)驗(yàn)證實(shí)內(nèi)皮祖細(xì)胞促進(jìn)內(nèi)皮細(xì)胞增殖和損傷后的的修復(fù)。促進(jìn)腦創(chuàng)傷后的血管修復(fù)蛋白表達(dá)水平增高,直接移植內(nèi)皮主細(xì)胞外泌體比移植干細(xì)胞更安全、有效,臨床應(yīng)用前景廣闊,但是仍需要對(duì)外泌體的基礎(chǔ)和臨床研究深入探討。
[Abstract]:Objective: traumatic brain injury (Traumaticbraininjury, TBI) serious harm to human health, with high morbidity and mortality. After craniocerebral trauma neurovascular repair decide the prognosis of patients, including repair of peripheral vascular trauma lesions is one of the key influencing factors. The subject of previous study confirmed that endothelial progenitor cells after traumatic brain injury (endothelialprogenitor cells, EPCs) mobilization increased, the trend xianyihouyang and in the peripheral blood, there is also evidence of endothelial progenitor cells capable of homing to the local traumatic lesions, promote traumatic lesions surrounding angiogenesis, but endothelial progenitor cells are directly differentiation into vascular endothelial cells and participate in angiogenesis at present is still not clear. Some reports proved that endothelial progenitor cells through paracrine action to promote the injury of the vascular stump of the endothelial cells to proliferate and differentiate to form new blood Tube, and not directly involved in the repair of differentiation into vascular endothelial cells. Endothelial progenitor cells have been shown to secrete exosomes, a variety of biological substances and endothelial progenitor cell exosomes contain the internal play a role, but the mechanism of nerve vascular repair exosomes derived endothelial progenitor cells in promoting after traumatic brain injury in it is not clear. In this experiment, through the observation of endothelial progenitor cells in exosomes in vivo endothelial progenitor cell exosomes to damage endothelial cells and its mechanism, to provide theoretical reference for the application of endothelial progenitor cell exosomes in treatment of traumatic brain injury. Methods: (1) from umbilical cord blood mononuclear cells in vitro the application of the method of density gradient centrifugation (MNCs), cultured and purified endothelial progenitor cells and identify its function; (2) by ultracentrifugation method obtained in vitro endothelial progenitor cell exosomes secreted into the culture medium, Application of transmission electron microscope, Westernblot method to observe the morphological characteristics of endothelial progenitor cells in exosomes and surface specific protein molecules. (3) by hypoxic endothelial cell injury model in vitro hypoxia box production method, application of Western blot detection of endothelial progenitor cell exosomes after intervention the expression changes of the tight junction protein expression and promote angiogenesis related proteins (4). The application of cerebral cortex injury instrument (CCI) preparation of craniocerebral injury in mice, puncture tail vein transplantation of endothelial progenitor cells exosomes, methods using immunofluorescence and Westernblot detection of endothelial progenitor cell transplantation exosomes after brain trauma wound around the focus of microvascular density changes the tight junction protein, blood brain barrier leakage of Evans blue, PCR was used to detect the tight junction protein, MMP9, expression of VEGF, application of Western blot technology to promote blood detection The expression of Guan Shengcheng protein PTEN. Results: the acquisition of umbilical cord blood mononuclear cells in vitro by gradient density centrifugation method successfully, and were cultured in vitro; exosome by ultracentrifugation method to obtain the source of endothelial progenitor cells by transmission electron microscopy, Westernblot method for identification of endothelial progenitor cells can secrete exosomes; for labeled with fluorescent dye PKH26 on the secretion of endothelial cells, endothelial progenitor cells can be confirmed phagocytosis of exosomes; in vitro endothelial progenitor cells promote injured endothelial cell tight junction protein CLN5, ZO-1, OCLN expression increased, PTEN expression decreased, increased the level of p-Akt. In vivo experiments suggested that umbilical cord blood endothelial progenitor cells can reduce into exosomes after traumatic brain injury of blood brain barrier permeability by Evans blue; PCR technology confirmed that traumatic lesions of tight junction protein CLN5, ZO-1, OCLN expression increased, VEGF expression level increased, MMP9 The expression level of Western increased; blot showed that exosomes can reduce the expression levels of third and seventh days PTEN traumatic lesions. Conclusion: endothelial progenitor cells can secrete exosomes of in vitro endothelial progenitor cells promote endothelial cell proliferation and repair after injury. To promote vascular repair protein expression after traumatic brain injury in the direct transplantation of endothelial cells and exosomes than transplanted stem cells more safe, effective, broad prospects of clinical application, but still need basic and clinical research on foreign body secretion in depth.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.15

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉德斌;石蓓;;間充質(zhì)干細(xì)胞來(lái)源外泌體的研究進(jìn)展[J];廣東醫(yī)學(xué);2017年01期

2 楊翔宇;李曉紅;肖靜;胡潔媚;馮娟;霍然;何國(guó)東;吳岳恒;余細(xì)勇;;干擾素γ刺激hUC-MSCs分泌外泌體促進(jìn)調(diào)節(jié)性T細(xì)胞生成[J];中國(guó)藥理學(xué)通報(bào);2017年01期

3 王鑫偉;毛建文;;試劑盒法與差速超速離心法所提血清外泌體的形態(tài)學(xué)比較[J];臨床與病理雜志;2016年11期

4 路康;楊匡;李海音;周躍;李長(zhǎng)青;;髓核細(xì)胞來(lái)源外泌體對(duì)骨髓間充質(zhì)干細(xì)胞向髓核樣細(xì)胞分化的作用[J];中國(guó)脊柱脊髓雜志;2016年10期

5 Etsu Suzuki;Daishi Fujita;Masao Takahashi;Shigeyoshi Oba;Hiroaki Nishimatsu;;Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease[J];World Journal of Stem Cells;2016年09期

6 李玲;石蓓;;干細(xì)胞源外泌體在治療心肌梗死中的作用機(jī)制研究進(jìn)展[J];醫(yī)學(xué)研究生學(xué)報(bào);2016年09期

7 王偉偉;艾開(kāi)興;;HiPSC-MSC外泌體促進(jìn)大鼠肝再生修復(fù)作用的研究[J];外科理論與實(shí)踐;2016年03期

8 宋菲;俞夢(mèng)越;;干細(xì)胞來(lái)源的外泌體:心肌梗死治療新啟示[J];心血管病學(xué)進(jìn)展;2016年02期

9 Gen Suzuki;;Translational research of adult stem cell therapy[J];World Journal of Cardiology;2015年11期

10 張娟;史晉叔;李劍;;間充質(zhì)干細(xì)胞源性外泌體——未來(lái)生物療法的理想載體[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2015年04期

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