調(diào)節(jié)性T細胞過繼轉(zhuǎn)輸減輕腦出血后血腦屏障損害和細胞凋亡
發(fā)布時間：2018-08-27 17:53
【摘要】：目的 建立小鼠腦出血（ICH）模型，將外源CD4+CD25+調(diào)節(jié)性T細胞經(jīng)過股靜脈過繼轉(zhuǎn)輸，探討調(diào)節(jié)性T細胞（CD4+CD25+Tregs）對出血后小鼠腦組織的保護作用和可能機制。 方法 實驗選取10-12周齡，體重25g左右的健康雄性昆明小鼠作為研究對象，將動物隨機分為：腦出血手術(shù)組和假手術(shù)對照（sham）組。采用改良的方式向小鼠左側(cè)紋狀體注入10ul的自體血制備腦出血模型；采用免疫磁珠法，提取小鼠脾臟內(nèi)CD4+CD25+Tregs；經(jīng)股靜脈輸注Tregs。腦出血手術(shù)組又分為三個亞組：Tregs組，輸注移植提取的CD4+CD25+Tregs；PBS組，輸注相同體積的PBS；ICH組：出血后未經(jīng)任何處理。采用角轉(zhuǎn)落、足失誤實驗等評分標準在術(shù)后一月內(nèi)對小鼠的神經(jīng)功能缺損進行評分。分別采用ELISA法（酶聯(lián)免疫吸附）、EB法（伊文思藍）、TUNEL（原位末端標記術(shù)）、Caspase-3（凋亡相關(guān)蛋白）對腦出血后自由基MDA及其清除劑SOD的變化，血腦屏障通透性，細胞凋亡等相關(guān)指標進行觀察。 結(jié)果 1.與假手術(shù)對照組相比，腦出血后1d各組小鼠便表現(xiàn)出不同程度的神經(jīng)功能缺損，尤以PBS組和ICH組最為嚴重（P＜0.05)，一個月后方恢復(fù)；Tregs組治療的角轉(zhuǎn)落及足失誤評分都顯著低于手術(shù)其他組（P＜0.05），3周時間基本恢復(fù)。 2.腦出血后免疫熒光顯微鏡觀察凋亡的有關(guān)指標可見：與假手術(shù)組比較，腦出血模型組小鼠血腫周圍組織的TUNEL、Caspase-3染色的細胞數(shù)增加，提示出現(xiàn)明顯的細胞凋亡（P＜0.05）。模型各亞組中又以ICH及PBS組細胞凋亡最為嚴重，Tregs組損傷最輕。與假手術(shù)組相比，小鼠腦出血后血腦屏障也遭破壞，，EB外滲量顯著升高，持續(xù)一周仍未好轉(zhuǎn)，具有顯著的統(tǒng)計學(xué)意義（P＜0.001)；腦出血組各時間點自由基MDA含量也顯著升高，自由基清除劑SOD的水平卻反向降低（P＜0.05）；Tregs組雖也出現(xiàn)類似變化，但程度較輕（P＜0.05）。 結(jié)論 1.采用改良的方式可成功制備小鼠腦出血模型，此模型能夠模擬ICH的主要病理和臨床過程。 2.外源性CD4+CD25+Tregs過繼轉(zhuǎn)輸對小鼠腦出血具有保護作用，可促進小鼠ICH后神經(jīng)功能的恢復(fù)。 3.外源性CD4+CD25+Tregs的過繼轉(zhuǎn)輸可保護小鼠腦出血后BBB的完整性。 4.外源性CD4+CD25+Tregs的過繼轉(zhuǎn)輸可以顯著降低腦出血后MDA的產(chǎn)生，減少SOD消耗，抑制細胞凋亡，減輕損傷，起到腦保護作用。
[Abstract]:Objective to establish (ICH) model of intracerebral hemorrhage in mice and transfer exogenous CD4 CD25 regulatory T cells through femoral vein to investigate the protective effect and possible mechanism of regulatory T cells (CD4 CD25 Tregs) on brain tissue of mice after intracerebral hemorrhage. Methods healthy male Kunming mice, 10-12 weeks old and weighing about 25g, were randomly divided into two groups: intracerebral hemorrhage operation group and sham operation control (sham) group. The model of intracerebral hemorrhage was established by injecting autogenous blood of 10ul into the left striatum of mice in a modified way, and CD4 CD25 Tregs; was extracted by immunomagnetic beads to infuse Tregs. through femoral vein in mice. The intracerebral hemorrhage operation group was divided into three subgroups: 1: Tregs group, infusion of CD4 CD25 Tregs;PBS extracted by transplantation, and infusion of the same volume of PBS;ICH: without any treatment after hemorrhage. The neurological deficits of mice were evaluated by the criteria of corner rotation and foot error test within one month after operation. ELISA (enzyme linked immunosorbent assay) EB method (Evans blue) and Tunel (in situ end labeling) and Caspase-3 (apoptosis-related protein) were used to observe the changes of free radical MDA and its scavenger SOD, blood-brain barrier permeability and apoptosis after intracerebral hemorrhage. Result 1. Compared with the sham-operated control group, the rats in each group showed different degrees of neurological impairment on the 1st day after ICH, especially in the PBS and ICH groups (P < 0. 05), and recovered after one month. In the Tregs group, the score of keratoplasty and foot error was significantly lower than that of the other groups (P < 0. 05). The relative indexes of apoptosis observed by immunofluorescence microscope after ICH were as follows: compared with the sham-operated group, the number of TUNEL,Caspase-3 staining cells around the hematoma in the ICH model group increased, indicating that there was obvious apoptosis (P < 0. 05). The apoptosis of ICH and PBS groups was the most serious and the damage of Tregs group was the least. Compared with the sham operation group, the exosmosis of EB in the blood brain barrier was significantly increased after ICH in mice, and it was not improved for one week (P < 0.001), and the content of free radical MDA in ICH group was also significantly higher than that in the control group (P < 0.05), and the content of free radical MDA in ICH group was also significantly higher than that in the control group. The level of free radical scavenger (SOD) decreased in the reverse direction (P < 0. 05), although the level of Tregs also showed similar changes, but the degree was less (P < 0. 05). Conclusion 1. The model of intracerebral hemorrhage in mice can be successfully prepared by using the improved method. This model can simulate the main pathological and clinical processes of ICH. 2. 2. Exogenous CD4 CD25 Tregs adoptive transfusions have protective effect on cerebral hemorrhage in mice and promote the recovery of nerve function after ICH. Adoptive transfusions of exogenous CD4 CD25 Tregs could protect the integrity of BBB after intracerebral hemorrhage in mice. 4. Adoptive transfusions of exogenous CD4 CD25 Tregs can significantly reduce the production of MDA, reduce the consumption of SOD, inhibit cell apoptosis, alleviate injury and play a protective role in brain after intracerebral hemorrhage.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.34
本文編號：2208000
[Abstract]:Objective to establish (ICH) model of intracerebral hemorrhage in mice and transfer exogenous CD4 CD25 regulatory T cells through femoral vein to investigate the protective effect and possible mechanism of regulatory T cells (CD4 CD25 Tregs) on brain tissue of mice after intracerebral hemorrhage. Methods healthy male Kunming mice, 10-12 weeks old and weighing about 25g, were randomly divided into two groups: intracerebral hemorrhage operation group and sham operation control (sham) group. The model of intracerebral hemorrhage was established by injecting autogenous blood of 10ul into the left striatum of mice in a modified way, and CD4 CD25 Tregs; was extracted by immunomagnetic beads to infuse Tregs. through femoral vein in mice. The intracerebral hemorrhage operation group was divided into three subgroups: 1: Tregs group, infusion of CD4 CD25 Tregs;PBS extracted by transplantation, and infusion of the same volume of PBS;ICH: without any treatment after hemorrhage. The neurological deficits of mice were evaluated by the criteria of corner rotation and foot error test within one month after operation. ELISA (enzyme linked immunosorbent assay) EB method (Evans blue) and Tunel (in situ end labeling) and Caspase-3 (apoptosis-related protein) were used to observe the changes of free radical MDA and its scavenger SOD, blood-brain barrier permeability and apoptosis after intracerebral hemorrhage. Result 1. Compared with the sham-operated control group, the rats in each group showed different degrees of neurological impairment on the 1st day after ICH, especially in the PBS and ICH groups (P < 0. 05), and recovered after one month. In the Tregs group, the score of keratoplasty and foot error was significantly lower than that of the other groups (P < 0. 05). The relative indexes of apoptosis observed by immunofluorescence microscope after ICH were as follows: compared with the sham-operated group, the number of TUNEL,Caspase-3 staining cells around the hematoma in the ICH model group increased, indicating that there was obvious apoptosis (P < 0. 05). The apoptosis of ICH and PBS groups was the most serious and the damage of Tregs group was the least. Compared with the sham operation group, the exosmosis of EB in the blood brain barrier was significantly increased after ICH in mice, and it was not improved for one week (P < 0.001), and the content of free radical MDA in ICH group was also significantly higher than that in the control group (P < 0.05), and the content of free radical MDA in ICH group was also significantly higher than that in the control group. The level of free radical scavenger (SOD) decreased in the reverse direction (P < 0. 05), although the level of Tregs also showed similar changes, but the degree was less (P < 0. 05). Conclusion 1. The model of intracerebral hemorrhage in mice can be successfully prepared by using the improved method. This model can simulate the main pathological and clinical processes of ICH. 2. 2. Exogenous CD4 CD25 Tregs adoptive transfusions have protective effect on cerebral hemorrhage in mice and promote the recovery of nerve function after ICH. Adoptive transfusions of exogenous CD4 CD25 Tregs could protect the integrity of BBB after intracerebral hemorrhage in mice. 4. Adoptive transfusions of exogenous CD4 CD25 Tregs can significantly reduce the production of MDA, reduce the consumption of SOD, inhibit cell apoptosis, alleviate injury and play a protective role in brain after intracerebral hemorrhage.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.34
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