体外扩增的调节性T细胞过继转输对缺血性脑卒中的保护作用及抗炎机制
发布时间：2018-08-17 11:06
【摘要】：实验目的： 1.建立大鼠CD4+CD25+调节性T细胞（regulatory T cells， Tregs）的分离和体外扩增方法，并检测分离的和扩增的Tregs纯度、活性及其免疫抑制活性。 2.探讨体外扩增的Tregs静脉输注移植对大鼠缺血性脑卒中的保护作用。 3.研究体外扩增的Tregs输注移植后脑缺血再灌注大鼠固有的和入侵的炎症细胞的变化，并探讨Tregs对脑缺血后神经炎症反应的抑制作用。 研究方法： 1.采用免疫磁珠细胞分选(Magnetic cell sorting, MACS)两步法从健康成年SD大鼠脾脏和淋巴结中提取CD4+CD25+Tregs，然后加入刺激因子anti-CD3、anti-CD28、IL-2以及雷帕霉素与之共培养进行体外扩增。用流式细胞仪测定分离的以及培养的细胞中Tregs的纯度，台盼蓝染色法检测细胞的活性，体外增殖抑制试验测定新鲜分离的和扩增的Tregs的增殖及其抑制功能。 2.线栓法制备右侧大脑中动脉阻塞（MCAO）模型，缺血2h再灌注。实验大鼠随机分成假手术组、PBS处理组、脾细胞（SP）处理组和Treg细胞治疗组。在Treg细胞输注后3d、7d、14d采用Zea Longa、触须诱导的前肢放置试验、足失误试验、圆筒实验对各组大鼠进行神经功能评分；于术后2w采用水迷宫实验评价各组大鼠的空间认知能力；行TTC染色和甲酚紫染色测量脑梗死体积比以及采用干湿称重法测量脑组织含水量；免疫荧光染色法检测缺血脑组织Caspase-3的表达情况；Fluro-JadeB染色观察神经元退化情况。 3.模型处理和分组同前。于Treg细胞输注后3d、14d采用免疫荧光和免疫组化染色观察MPO、Iba1、GFAP在脑组织的表达情况以检测Treg细胞输注后对中性粒细胞（MPO）向脑部的浸润以及脑固有细胞如小胶质细胞（Iba1）和星形胶质细胞（GFAP）的反应性的影响，并采用Werstern Blot方法进一步验证各组大鼠脑组织中Iba1、GFAP的表达情况。 结果： 1.MACS分选获得的CD4+CD25+regulatory T细胞的纯度是84.50±5.08%，细胞存活率为95.22±2.97%。经过3周体外培养扩增，Tregs的纯度为76.03±4.04%，，细胞存活率为94.31±3.14%。体外增殖抑制实验表明Tregs能显著抑制CD4+CD25-T细胞的增殖（P＜0.01），体外扩增的Tregs的抑制功能超过新鲜分离的细胞，差异具有统计学意义（P＜0.05）。 2.脑缺血再灌注后3d，模型组大鼠各项神经功能评分达到高峰，与PBS组和SP组相比Tregs治疗组的感觉运动功能得到显著改善。Morris Water Maze test显示在MCAO后14d，Tregs治疗组的空间学习和记忆能力较PBS组和SP组均有显著改善。TTC染色和甲酚紫染色结果显示在MCAO后不同时间点Treg治疗组的脑梗死体积较PBS和SP处理组均显著降低。在MCAO后不同时间点，Tregs治疗组的脑组织含水量较PBS和SP处理组均有明显改善；Caspase-3、Fluro-JadeB染色结果显示Tregs治疗组脑缺血半暗带区的阳性细胞数目较PBS和SP处理组显著降低。 3.免疫荧光染色观察脑组织中中性粒细胞的浸润情况，结果显示假手术组无或可见少量散在分布的MPO+细胞，PBS组和SP组MPO+细胞在缺血再灌注1-3d时向脑部浸润明显，Tregs治疗组的MPO+细胞数显著降低。免疫组化和免疫荧光染色结果显示在脑缺血2w时Tregs治疗组Iba1阳性和GFAP阳性细胞数较PBS和SP处理组显著降低，WesternBlot结果与免疫组化结果一致。 结论 1.本实验建立的分离和扩增大鼠CD4+CD25+调节性T细胞的方法，可有效的获得高纯度、有活力且不影响其抑制功能的Treg细胞。 2.扩增的CD4+CD25+Tregs治疗性输注可显著降低大脑中动脉阻塞脑缺血再灌注后脑梗死体积，降低凋亡及退化的神经元数目，并显著改善脑缺血再灌注后的神经功能损伤。 3.体外培养扩增的Tregs治疗性输注可能是通过抑制外周中性粒细胞浸润以及调节小胶质细胞和星形胶质细胞的活化来介导Tregs对脑缺血再灌注的保护作用。
[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.
【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】：R743.3
本文编号：2187407
[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.
【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】：R743.3
【参考文献】
相关期刊论文 前2条
1 黄立锋;姚咏明;董宁;张立天;盛志勇;;大鼠CD4~+CD25~+调节性T细胞的分离及功能鉴定[J];感染.炎症.修复;2008年03期
2 史艳侠;何伟;彭柔君;姜文奇;;CD4~+CD25~+调节性T细胞的体外扩增[J];中山大学学报(医学科学版);2008年06期
本文编号：2187407
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