调节性T细胞过继转输减轻脑出血后血脑屏障损害和细胞凋亡
发布时间：2018-08-27 17:53
【摘要】：目的 建立小鼠脑出血（ICH）模型，将外源CD4+CD25+调节性T细胞经过股静脉过继转输，探讨调节性T细胞（CD4+CD25+Tregs）对出血后小鼠脑组织的保护作用和可能机制。 方法 实验选取10-12周龄，体重25g左右的健康雄性昆明小鼠作为研究对象，将动物随机分为：脑出血手术组和假手术对照（sham）组。采用改良的方式向小鼠左侧纹状体注入10ul的自体血制备脑出血模型；采用免疫磁珠法，提取小鼠脾脏内CD4+CD25+Tregs；经股静脉输注Tregs。脑出血手术组又分为三个亚组：Tregs组，输注移植提取的CD4+CD25+Tregs；PBS组，输注相同体积的PBS；ICH组：出血后未经任何处理。采用角转落、足失误实验等评分标准在术后一月内对小鼠的神经功能缺损进行评分。分别采用ELISA法（酶联免疫吸附）、EB法（伊文思蓝）、TUNEL（原位末端标记术）、Caspase-3（凋亡相关蛋白）对脑出血后自由基MDA及其清除剂SOD的变化，血脑屏障通透性，细胞凋亡等相关指标进行观察。 结果 1.与假手术对照组相比，脑出血后1d各组小鼠便表现出不同程度的神经功能缺损，尤以PBS组和ICH组最为严重（P＜0.05)，一个月后方恢复；Tregs组治疗的角转落及足失误评分都显著低于手术其他组（P＜0.05），3周时间基本恢复。 2.脑出血后免疫荧光显微镜观察凋亡的有关指标可见：与假手术组比较，脑出血模型组小鼠血肿周围组织的TUNEL、Caspase-3染色的细胞数增加，提示出现明显的细胞凋亡（P＜0.05）。模型各亚组中又以ICH及PBS组细胞凋亡最为严重，Tregs组损伤最轻。与假手术组相比，小鼠脑出血后血脑屏障也遭破坏，，EB外渗量显著升高，持续一周仍未好转，具有显著的统计学意义（P＜0.001)；脑出血组各时间点自由基MDA含量也显著升高，自由基清除剂SOD的水平却反向降低（P＜0.05）；Tregs组虽也出现类似变化，但程度较轻（P＜0.05）。 结论 1.采用改良的方式可成功制备小鼠脑出血模型，此模型能够模拟ICH的主要病理和临床过程。 2.外源性CD4+CD25+Tregs过继转输对小鼠脑出血具有保护作用，可促进小鼠ICH后神经功能的恢复。 3.外源性CD4+CD25+Tregs的过继转输可保护小鼠脑出血后BBB的完整性。 4.外源性CD4+CD25+Tregs的过继转输可以显著降低脑出血后MDA的产生，减少SOD消耗，抑制细胞凋亡，减轻损伤，起到脑保护作用。
[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.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34
本文编号：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.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34
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