發(fā)布時間：2018-04-16 04:12

  本文選題：腦梗死 + 脂肪間充質(zhì)干細(xì)胞�。� 參考：《吉林大學(xué)》2017年博士論文

【摘要】：研究背景:腦梗死是神經(jīng)內(nèi)科的常見病,多發(fā)病。在世界范圍內(nèi),它是導(dǎo)致死亡和致殘的第二位原因。而在中國,它是導(dǎo)致死亡和成人致殘的第一位原因。每年,至少有150萬新發(fā)的腦梗死患者,他們中將近30%遺留有持久的嚴(yán)重的殘疾。腦梗死已經(jīng)是社會和家庭的主要負(fù)擔(dān)。靜脈溶栓和機(jī)械取栓是目前僅有的被FDA批準(zhǔn)的急性腦梗死的治療方法。但是,由于時間窗較短和有出血性轉(zhuǎn)化等風(fēng)險,僅有少數(shù)的患者能從中受益。因此,迫切需要一個新的療法來治療腦梗死。干細(xì)胞是近幾年臨床研究的熱點。尤其是間充質(zhì)干細(xì)胞,大量基礎(chǔ)實驗均證實了間充質(zhì)干細(xì)胞的安全性和有效性,因此現(xiàn)已被批準(zhǔn)用于臨床研究。間充質(zhì)干細(xì)胞科來源于骨髓、脂肪、臍帶血等。雖然骨髓間充質(zhì)干細(xì)胞是目前研究和應(yīng)用最多的細(xì)胞,但是,實驗表明,脂肪間充質(zhì)干細(xì)胞和骨髓間充質(zhì)干細(xì)胞具有相同的生物學(xué)特性,而且,脂肪間充質(zhì)干細(xì)胞比骨髓間充質(zhì)干細(xì)胞具有更多的優(yōu)勢,它具有來源豐富,取材簡單,易于培養(yǎng)等特點。在缺血性腦卒中治療中,具有調(diào)節(jié)炎癥、血管再生及抗凋亡等作用,能夠明顯的改善血腦屏障的通透性,減少梗死體積,改善神經(jīng)功能缺損癥狀。因此,脂肪間充質(zhì)干細(xì)胞移植治療有望成為臨床上治療急性腦梗死的最為理想的細(xì)胞。越來越多的證據(jù)表明,脂肪間充質(zhì)干細(xì)胞能夠改善缺血再灌注損傷大鼠的神經(jīng)功能,但是,其作用機(jī)制一直比較復(fù)雜。了解其作用機(jī)制有助于基礎(chǔ)向臨床的轉(zhuǎn)化。目前的研究表明,脂肪間充質(zhì)干細(xì)胞在腦梗死的治療中具有抗炎、抗凋亡、促進(jìn)血管再生、神經(jīng)再生等作用。尤其是急性期,抗炎抗凋亡的作用尤為顯著。大量研究炎癥與缺血再灌注損傷后的血腦屏障的破壞是密不可分的。未折疊蛋白反應(yīng)(UPR)可以促進(jìn)腦梗死后的細(xì)胞凋亡。因此,本實驗選用ADMSCs用于治療急性腦梗死大鼠,并通過對炎癥介質(zhì)、凋亡反應(yīng)、血腦屏障透通性、UPR調(diào)節(jié)的觀察來探討ADMSCs可能的作用機(jī)制。目的:(1)觀察ADMSCs的表面標(biāo)志物及其向脂肪細(xì)胞和成骨細(xì)胞的定向分化能力;(2)觀察ADMSCs治療后血腦屏障的變化;(3)觀察ADMCs對缺血再灌注大鼠腦梗死體積的影響及其對神經(jīng)功能缺損癥狀的影響;(4)觀察ADMSCs對腦梗死后小膠質(zhì)細(xì)胞增生和炎癥介質(zhì)釋放的影響;(5)觀察ADMSCs對凋亡及UPR調(diào)節(jié)的影響。根據(jù)這些結(jié)果進(jìn)一步證實ADMSCs對急性腦梗死治療的有效性及其作用機(jī)制。實驗方法:(1)選取250g-300g的SD大鼠作為實驗對象,制作大腦中動脈閉塞(MCAO)-2小時缺血再灌注模型;(2)提取分離并培養(yǎng)脂肪間充質(zhì)干細(xì)胞,選用P3代細(xì)胞作為實驗用細(xì)胞,在細(xì)胞注射前30分鐘,用流式細(xì)胞儀檢測法檢測ADMSCs的表面標(biāo)志物。同時,將P3代細(xì)胞用成脂和成骨誘導(dǎo)液進(jìn)行誘導(dǎo),21天后采用油紅O染色和茜素紅染色的方法鑒定ADMSCs向脂肪細(xì)胞和成骨細(xì)胞的分化能力;(3)將MCAO大鼠隨機(jī)分為3組,sham組,MCAO組和MCAO+ADMSCs組,在造模后0,12小時,24小時分別給予尾靜脈注射等容量PBS和ADMSCs(2×106個);(4)治療后第1天,第3天,第7天,第14天用m NSS評分法進(jìn)行大鼠的行為功能測試;(5)在治療第1天,第7天,第14天用伊文思藍(lán)染色的方法觀察大鼠血腦屏障的變化;(6)在治療第14天處死大鼠,通過免疫熒光染色觀察大鼠血腦屏障緊密連接蛋白ZO-1和Claudin-5的表達(dá),同時觀察小膠質(zhì)細(xì)胞增生OX-42的表達(dá);(7)在治療14天,用PT-PCR技術(shù)檢測炎癥介質(zhì)TNF-α,IL-1β,IL-6的表達(dá);(8)通過TUNEL染色的方法觀察梗死區(qū)神經(jīng)細(xì)胞的凋亡;(9)通過westernblot的方法觀察ADMSCs對UPR調(diào)節(jié)信號通路的GRP78,PERK,p-PERK,e IF2α,p-e IF2α,ATF4,CHOP,Bax,Bcl-2,XBP-1表達(dá)的影響。實驗結(jié)果:(1)ADMSCs高度表達(dá)間充質(zhì)干細(xì)胞的表面標(biāo)志物CD44,CD29,CD90,而不表達(dá)造血干細(xì)胞的表面抗原CD34,CD45;(2)成脂誘導(dǎo)液和成骨誘導(dǎo)液誘導(dǎo)21天后,油紅O染色和茜素紅染色發(fā)現(xiàn)ADMSCs可以向成脂細(xì)胞和成骨細(xì)胞定向分化;(3)TTC染色觀察到,與MCAO相比,ADMSCs治療能明顯的減少M(fèi)CAO大鼠的梗死體積(P0.01);(4)m NSS評分的比較,ADMSCs組明顯的降低m NSS的評分,這種改善神經(jīng)功能缺損的效果從第7天開始一直持續(xù)到第14天(P0.01);(5)伊文思藍(lán)染色顯示,造模后第一天開始血腦屏障明顯破壞,并隨著時間的延長逐漸好轉(zhuǎn),ADMSCs治療在第7天明顯改善MCAO引起的血腦屏障的破壞;(6)PT-PCR檢測發(fā)現(xiàn),MCAO后梗死區(qū)的TNF-α,IL-1β,IL-6的表達(dá)明顯增高(與sham組相比,P0.01),ADMSCs治療能夠明顯的降低這三種炎癥介質(zhì)的釋放(P0.01);(7)免疫熒光染色發(fā)現(xiàn),MCAO組血腦屏障愛緊密連接蛋白ZO-1,Claudin-5的熒光強(qiáng)度明顯降低(P0.01),ADMSCs治療后能明顯的增強(qiáng)它們熒光強(qiáng)度的表達(dá)(P0.01),說明ADMSCs能改善缺血再灌注損傷的血腦屏障;(8)免疫熒光染色同時發(fā)現(xiàn)ADMSCs治療能減少M(fèi)CAO后的OX-42的表達(dá),兩組之間具有顯著差異(P0.01);(9)ADMSCs治療能夠明顯的減少腦梗死及周邊區(qū)域的細(xì)胞凋亡,與MCAO組具有顯著差異(P0.01);(10)通過Westernblot觀察UPR信號通路上的蛋白,發(fā)現(xiàn)ADMSCs能夠明顯的升高抗凋亡因子Bcl-2的表達(dá),降低GRP78,p-PERK,p-e IF2α,ATF4,CHOP,Bax,XBP的表達(dá),與MCAO組相比具有顯著的統(tǒng)計學(xué)差異(P0.01)。結(jié)論:(1)ADMSCs取材簡單,來源豐富,易于培養(yǎng),沒有免疫排斥反應(yīng),具有誘導(dǎo)分化的能力,是基礎(chǔ)及臨床上細(xì)胞治療的較為理想的細(xì)胞來源。(2)多次、靜脈注射ADMSCs能夠明顯的減少腦缺血再灌注損傷的梗死體積,改善神經(jīng)功能缺損癥狀。(3)早期給予ADMSCs能夠明顯的改善缺血再灌注損傷大鼠的血腦屏障。(4)ADMSCs治療能夠減輕缺血再灌注大鼠的炎癥反應(yīng),進(jìn)而降低血腦屏障的通透性。(5)腦缺血再灌注損傷能夠誘發(fā)內(nèi)質(zhì)網(wǎng)的UPR,促進(jìn)凋亡反應(yīng)。ADMSCs可能是通過調(diào)節(jié)UPR反應(yīng)來降低凋亡,進(jìn)而改善MCAO大鼠的神經(jīng)功能癥狀。
[Abstract]:Background: cerebral infarction is a common disease in neurology, the disease. In the world, it is the leading cause of death and disability in second. China, it is leading cause of death and disability in adults. Every year, there are at least 150 million new cases of cerebral infarction patients, they will have a lasting legacy of nearly 30% the serious disability. Cerebral infarction is a major burden on society and family. Intravenous thrombolysis and mechanical thrombectomy is the treatment of acute cerebral infarction is currently the only approved by the FDA. However, due to a relatively short time window and the risk of hemorrhagic transformation, only a small number of patients can benefit from it. Therefore, an urgent the new therapy to treatment of cerebral infarction. Stem cells are the hotspot in recent years. Especially the clinical study of mesenchymal stem cells, a large number of basic experiments have confirmed the safety and efficacy of mesenchymal stem cells, so it has been approved by In the clinical study. Mesenchymal stem cells derived from bone marrow, umbilical cord blood, fat, etc.. Although bone marrow mesenchymal stem cells is the research and application of most cells, however, experiments show that adipose derived mesenchymal stem cells and bone marrow mesenchymal stem cells have the same biological characteristics, but also between fat mesenchymal stem cells have more advantages than the bone marrow mesenchymal stem cells, it has a rich source of material is simple, easy to culture and so on. In the treatment of ischemic stroke, with the regulation of inflammation, angiogenesis and anti apoptosis, can significantly improve the permeability of the blood-brain barrier, reduce infarct volume and improve defect the symptoms of nerve function. Therefore, adipose derived mesenchymal stem cells transplantation for the treatment of the clinical treatment of acute cerebral infarction is expected to become the most ideal cells. More and more evidence that adipose derived mesenchymal stem cells can improve ischemia reperfusion Note the damage of neurological function in rats, but its mechanism is complex. To investigate the mechanism of help to clinical translation. The present study showed that adipose derived mesenchymal stem cells in the treatment of cerebral infarction with anti-inflammatory, anti apoptosis, promoting angiogenesis, nerve regeneration and so on. Especially acute, anti-inflammatory and anti apoptosis effect is particularly significant. A lot of inflammation and ischemia reperfusion injury after the destruction of the blood-brain barrier is inseparable. The unfolded protein response (UPR) can promote cell apoptosis after cerebral infarction. Therefore, this experiment used ADMSCs for the treatment of acute cerebral infarction in rats, and the apoptosis in response to inflammation medium, blood brain barrier permeability, observe the regulation of UPR to explore the possible mechanisms of ADMSCs. Objective: (1) to observe the surface markers of ADMSCs were related to adipogenic and osteogenic differentiation ability; (2) The changes of blood brain barrier was observed after treatment with ADMSCs; (3) effect of ADMCs cerebral infarction volume in rats on ischemia reperfusion and its effect on neurological function; (4) to observe the effect of ADMSCs on proliferation of microglia and release of inflammatory mediators after cerebral infarction; (5) to observe the effect of ADMSCs on apoptosis and regulation UPR. According to these results further confirmed the effectiveness of ADMSCs for the treatment of acute cerebral infarction and its mechanism. Methods: (1) select 250g-300g SD rats as experimental object, making middle cerebral artery occlusion (MCAO) -2 hour ischemia reperfusion model; (2) adipose derived mesenchymal stem cells and culture extraction the P3 generation of cells, as in the experiment, cells were injected 30 minutes before using the surface markers of ADMSCs by flow cytometry assay. At the same time, the fat and osteogenic induction medium in induced by P3 cells, after 21 days by oil red O 鏌撹壊鍜岃寽绱犵孩鏌撹壊鐨勬柟娉曢壌瀹欰DMSCs鍚戣剛鑲粏鑳?yōu)鍜屾垚楠ňl嗚優(yōu)鐨勫垎鍖栬兘鍔，

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