【摘要】：實(shí)驗(yàn)?zāi)康模?1.建立大鼠CD4+CD25+調(diào)節(jié)性T細(xì)胞（regulatory T cells， Tregs）的分離和體外擴(kuò)增方法，并檢測分離的和擴(kuò)增的Tregs純度、活性及其免疫抑制活性。 2.探討體外擴(kuò)增的Tregs靜脈輸注移植對(duì)大鼠缺血性腦卒中的保護(hù)作用。 3.研究體外擴(kuò)增的Tregs輸注移植后腦缺血再灌注大鼠固有的和入侵的炎癥細(xì)胞的變化，并探討Tregs對(duì)腦缺血后神經(jīng)炎癥反應(yīng)的抑制作用。 研究方法： 1.采用免疫磁珠細(xì)胞分選(Magnetic cell sorting, MACS)兩步法從健康成年SD大鼠脾臟和淋巴結(jié)中提取CD4+CD25+Tregs，然后加入刺激因子anti-CD3、anti-CD28、IL-2以及雷帕霉素與之共培養(yǎng)進(jìn)行體外擴(kuò)增。用流式細(xì)胞儀測定分離的以及培養(yǎng)的細(xì)胞中Tregs的純度，臺(tái)盼藍(lán)染色法檢測細(xì)胞的活性，體外增殖抑制試驗(yàn)測定新鮮分離的和擴(kuò)增的Tregs的增殖及其抑制功能。 2.線栓法制備右側(cè)大腦中動(dòng)脈阻塞（MCAO）模型，缺血2h再灌注。實(shí)驗(yàn)大鼠隨機(jī)分成假手術(shù)組、PBS處理組、脾細(xì)胞（SP）處理組和Treg細(xì)胞治療組。在Treg細(xì)胞輸注后3d、7d、14d采用Zea Longa、觸須誘導(dǎo)的前肢放置試驗(yàn)、足失誤試驗(yàn)、圓筒實(shí)驗(yàn)對(duì)各組大鼠進(jìn)行神經(jīng)功能評(píng)分；于術(shù)后2w采用水迷宮實(shí)驗(yàn)評(píng)價(jià)各組大鼠的空間認(rèn)知能力；行TTC染色和甲酚紫染色測量腦梗死體積比以及采用干濕稱重法測量腦組織含水量；免疫熒光染色法檢測缺血腦組織Caspase-3的表達(dá)情況；Fluro-JadeB染色觀察神經(jīng)元退化情況。 3.模型處理和分組同前。于Treg細(xì)胞輸注后3d、14d采用免疫熒光和免疫組化染色觀察MPO、Iba1、GFAP在腦組織的表達(dá)情況以檢測Treg細(xì)胞輸注后對(duì)中性粒細(xì)胞（MPO）向腦部的浸潤以及腦固有細(xì)胞如小膠質(zhì)細(xì)胞（Iba1）和星形膠質(zhì)細(xì)胞（GFAP）的反應(yīng)性的影響，并采用Werstern Blot方法進(jìn)一步驗(yàn)證各組大鼠腦組織中Iba1、GFAP的表達(dá)情況。 結(jié)果： 1.MACS分選獲得的CD4+CD25+regulatory T細(xì)胞的純度是84.50±5.08%，細(xì)胞存活率為95.22±2.97%。經(jīng)過3周體外培養(yǎng)擴(kuò)增，Tregs的純度為76.03±4.04%，，細(xì)胞存活率為94.31±3.14%。體外增殖抑制實(shí)驗(yàn)表明Tregs能顯著抑制CD4+CD25-T細(xì)胞的增殖（P＜0.01），體外擴(kuò)增的Tregs的抑制功能超過新鮮分離的細(xì)胞，差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 2.腦缺血再灌注后3d，模型組大鼠各項(xiàng)神經(jīng)功能評(píng)分達(dá)到高峰，與PBS組和SP組相比Tregs治療組的感覺運(yùn)動(dòng)功能得到顯著改善。Morris Water Maze test顯示在MCAO后14d，Tregs治療組的空間學(xué)習(xí)和記憶能力較PBS組和SP組均有顯著改善。TTC染色和甲酚紫染色結(jié)果顯示在MCAO后不同時(shí)間點(diǎn)Treg治療組的腦梗死體積較PBS和SP處理組均顯著降低。在MCAO后不同時(shí)間點(diǎn)，Tregs治療組的腦組織含水量較PBS和SP處理組均有明顯改善；Caspase-3、Fluro-JadeB染色結(jié)果顯示Tregs治療組腦缺血半暗帶區(qū)的陽性細(xì)胞數(shù)目較PBS和SP處理組顯著降低。 3.免疫熒光染色觀察腦組織中中性粒細(xì)胞的浸潤情況，結(jié)果顯示假手術(shù)組無或可見少量散在分布的MPO+細(xì)胞，PBS組和SP組MPO+細(xì)胞在缺血再灌注1-3d時(shí)向腦部浸潤明顯，Tregs治療組的MPO+細(xì)胞數(shù)顯著降低。免疫組化和免疫熒光染色結(jié)果顯示在腦缺血2w時(shí)Tregs治療組Iba1陽性和GFAP陽性細(xì)胞數(shù)較PBS和SP處理組顯著降低，WesternBlot結(jié)果與免疫組化結(jié)果一致。 結(jié)論 1.本實(shí)驗(yàn)建立的分離和擴(kuò)增大鼠CD4+CD25+調(diào)節(jié)性T細(xì)胞的方法，可有效的獲得高純度、有活力且不影響其抑制功能的Treg細(xì)胞。 2.擴(kuò)增的CD4+CD25+Tregs治療性輸注可顯著降低大腦中動(dòng)脈阻塞腦缺血再灌注后腦梗死體積，降低凋亡及退化的神經(jīng)元數(shù)目，并顯著改善腦缺血再灌注后的神經(jīng)功能損傷。 3.體外培養(yǎng)擴(kuò)增的Tregs治療性輸注可能是通過抑制外周中性粒細(xì)胞浸潤以及調(diào)節(jié)小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞的活化來介導(dǎo)Tregs對(duì)腦缺血再灌注的保護(hù)作用。
[Abstract]:Objective:
1. To establish a method for isolation and in vitro amplification of rat CD4 + CD25 + regulatory T cells (Tregs), and to detect the purity, activity and immunosuppressive activity of isolated and amplified Tregs.
2. to explore the protective effect of intravenous infusion of Tregs on ischemic stroke in rats.
3. To study the changes of intrinsic and invasive inflammatory cells in rats with cerebral ischemia-reperfusion after in vitro amplified Tregs infusion transplantation, and to explore the inhibitory effect of Tregs on neuroinflammation after cerebral ischemia-reperfusion.
Research methods:
1. CD4+CD25+Tregs were extracted from spleen and lymph nodes of healthy adult SD rats by two-step immunomagnetic cell sorting (MACS) method, and then co-cultured with anti-CD3, anti-CD28, IL-2 and rapamycin for in vitro amplification. Purity, Trypan blue staining and in vitro proliferation inhibition test were used to determine the proliferation and inhibitory function of freshly isolated and amplified Tregs.
2. The right middle cerebral artery occlusion (MCAO) model was established by thread embolization and ischemia-reperfusion for 2 hours. The rats were randomly divided into sham-operated group, PBS group, SP group and Treg cell group. Neurological function score, water maze test, TTC staining, cresol violet staining, dry-wet weighing, immunofluorescence staining, Caspase-3 expression in ischemic brain tissue, and fluro-JadeB staining were used to evaluate the spatial cognitive ability of rats. Neuronal degeneration.
3. Immunofluorescence and immunohistochemical staining were used to observe the expression of MPO, Iba1 and GFAP in the brain tissues on the 3rd and 14th day after Treg cell infusion to detect the infiltration of neutrophils (MPO) into the brain and the responsiveness of intrinsic brain cells such as microglia (Iba1) and astrocytes (GFAP) after Treg cell infusion. Werstern Blot was used to further verify the expression of Iba1 and GFAP in brain tissue of each group.
Result:
1. The purity and survival rate of CD4+CD25+regulatory T cells were 84.50+5.08% and 95.22+2.97% respectively. The purity of Tregs was 76.03+4.04% and the cell survival rate was 94.31+3.14% after 3 weeks of culture and amplification in vitro. The proliferation inhibition experiment in vitro showed that Tregs could significantly inhibit the proliferation of CD4+CD25-T cells (P < 0.01). The inhibitory effect of S was greater than that of fresh cells, and the difference was statistically significant (P < 0.05).
2. Three days after cerebral ischemia and reperfusion, the neurological function scores of the model group reached the peak, and the sensorimotor function of the Tregs group was significantly improved compared with that of the PBS group and SP group. Morris Water Maze test showed that the spatial learning and memory abilities of the Tregs group were significantly improved 14 days after MCAO compared with those of the PBS group and SP group. The results of staining showed that the volume of cerebral infarction in Treg treatment group was significantly lower than that in PBS and SP treatment group at different time points after MCAO. At different time points after MCAO, the water content of brain tissue in Tregs treatment group was significantly improved compared with that in PBS and SP treatment group; Caspase-3 and Fluro-JadeB staining showed that the positive area of cerebral ischemic penumbra in Tregs treatment group was fine. The number of cells was significantly lower than that of PBS and SP treated groups.
3. The infiltration of neutrophils in brain tissue was observed by immunofluorescence staining. The results showed that there were no or few MPO + cells scattered in the sham operation group. MPO + cells in PBS group and SP group infiltrated into the brain significantly at 1-3 days after ischemia-reperfusion, and the number of MPO + cells in Tregs group decreased significantly. The number of Iba1-positive and GFAP-positive cells in Tregs treatment group was significantly lower than that in PBS and SP treatment groups at 2 weeks after cerebral ischemia. The results of Western Blot were consistent with the results of immunohistochemistry.
conclusion
1. The method of isolating and enlarging rat CD4+CD25+ regulatory T cells was established in this study. Treg cells with high purity, viability and no effect on their inhibitory function were obtained.
2. Amplified CD4+CD25+Tregs therapeutic infusion can significantly reduce the volume of cerebral infarction after middle cerebral artery occlusion and cerebral ischemia-reperfusion, reduce the number of apoptotic and degenerative neurons, and significantly improve the neurological impairment after cerebral ischemia-reperfusion.
3. Therapeutic infusion of cultured and amplified Tregs may mediate the protective effect of Tregs on cerebral ischemia-reperfusion by inhibiting the infiltration of peripheral neutrophils and regulating the activation of microglia and astrocytes.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 黃立鋒;姚詠明;董寧;張立天;盛志勇;;大鼠CD4~+CD25~+調(diào)節(jié)性T細(xì)胞的分離及功能鑒定[J];感染.炎癥.修復(fù);2008年03期

2 史艷俠;何偉;彭柔君;姜文奇;;CD4~+CD25~+調(diào)節(jié)性T細(xì)胞的體外擴(kuò)增[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2008年06期

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