内源性调节性T细胞在蛛网膜下腔血后脑血管痉挛及脑损伤中的作用

发布时间：2018-05-15 21:35

本文选题：内源性调节性T细胞 + 蛛网膜下腔出血　；参考：《泰山医学院》2014年硕士论文

【摘要】：研究目的 1.探讨内源性CD4+CD25+调节性T细胞删除后对蛛网膜下腔出血后小鼠神经功能的影响。 2.探讨内源性调节性T细胞删除对蛛网膜下腔出血后早期脑损伤的影响。 3.通过酶联免疫吸附法测定皮层IL-6、 TNF-α,初步探讨CD4+CD25+调节性T细胞删除对脑组织炎症的影响。 4.通过免疫荧光法检测皮层区炎症因子IL-6、GFAP的含量，通过westernblot检测脑组织中IL-6的含量，进一步探讨内源性CD4+CD25+调节性T细胞删除对蛛网膜下腔出血后脑组织炎症的影响。研究方法将雄性昆明小鼠（25-30g），随机分入假手术组、SAH组、Tregs删除组以及PBS注射组中。用血管内穿刺法建立小鼠蛛网膜下腔出血模型，激光多普勒血流仪记录顶叶皮层脑组织血流量的变化。假手术组不穿破血管。Tregs删除组模型需经过小鼠腹腔注射0.20mgCD25+特异性抗体（PC61mAb），来构建Treg细胞缺失小鼠。PBS腹腔注射组，经腹腔注射0.20mg的磷酸盐缓冲液。术后处死小鼠后,快速取脑,并用照相机拍摄脑基底池部出血情况,根据Sugawara等的报道,进行出血量评分,判定出血等级。采用Garcia评分系统对运动感觉进行评分,总分为18分。于蛛网膜下腔出血后72h，，用干湿重比较法检测脑组织含水率。用伊文思蓝法评价各组动物血脑屏障通透性。于术后72h，取脑制成石蜡切片，行苏木素-伊红染色，于镜下观察各组小鼠基底动脉管径大小以及管壁厚度的变化。制作脑切片后尼氏染色观察神经细胞变化。用TUNEL染色观察脑部细胞凋亡。用免疫荧光法测定皮层IL6、GFAP的表达及分布情况。用酶联免疫吸附法测定脑组织中IL-6、TNF-α含量。用western blot检测皮层组织炎症因子IL-6的含量。研究结果 1.出血程度：假手术组无出血，假手术组无出血；蛛网膜下腔出血组、PBS注射组、Tregs删除组在蛛网膜下腔均见到血液，各组之间出血量无统计意义的差异。 2.神经功能评分：假手术组评分为18分， SAH组、 PBS注射组与假手术组评分相比，有统计学意义(P0.05)，Tregs删除组与PBS注射组评分相比，有统计学意义(P0.05)，与SAH组的评分相比也有统计学意义(P0.05)。 3.血脑屏障通透性及脑水肿测定：小鼠SAH后血脑屏障通透性增加，脑组织含水量加重；Tregs删除组与SAH组相比，血脑屏障通透性显著增加(P0．05)，脑组织含水量显著加重(P0.05)。 4.基底动脉形态：小鼠蛛网膜下腔出血后基底动脉明显痉挛，Tregs删除后痉挛更为显著(P0.05)。 5.尼氏染色：蛛网膜下腔出血后，小鼠皮层中尼氏小体的神经元数量明显减少，Tregs删除后减少更为显著(P0.05)。 6.细胞凋亡检测：蛛网膜下腔出血后，小鼠皮层区凋亡细胞数增多，Tregs删除后与SAH后相比,有显著统计学以及意义（P0.05) 7.免疫荧光法测定皮层区IL-6、 GFAP SAH组、 PBS注射组、 Treg细胞删除组皮层区表达量均增多，Tregs删除组增多更为显著(P0．05)。 8.ELISA测定脑组织中IL-6和TNF-α含量： SAH后小鼠脑组织中IL-6和TNF-α表达量均有所升高， Tregs删除组与之相比更显著(P0.05) 9.Western blot检测皮层区IL-6： SAH组、 PBS组、 Tregs删除组的脑组织中IL-6表达量均增多，Tregs删除组与SAH组、假手术组相比有显著统计学意义(P0.05)。研究结论 1.本研究成功构建了蛛网膜下腔出血小鼠模型以及Tregs删除小鼠模型，并发现Tregs删除后会加重脑血管痉挛、脑组织水肿、血脑屏障通透性、凋亡等，同时也加重脑组织的炎性损伤。 2.小鼠SAH后产生神经功能有所损害，Tregs删除后可加重小鼠运动、感觉功能的损伤。
[Abstract]:research objective
1. to investigate the effect of deletion of endogenous CD4+CD25+ regulatory T cells on neurological function after subarachnoid hemorrhage in mice.
2. to investigate the effect of endogenous regulatory T cell deletion on early brain injury after subarachnoid hemorrhage.
3. to detect cortical IL-6 and TNF- alpha by enzyme-linked immunosorbent assay (ELISA), and preliminarily investigate the effect of CD4+CD25+ regulatory T cell deletion on brain tissue inflammation.
4. the content of inflammatory factors IL-6, GFAP, and the content of IL-6 in brain tissue were detected by immunofluorescence, and the effect of endogenous CD4+CD25+ regulatory T cell deletion on the brain tissue inflammation after subarachnoid hemorrhage was further investigated.
research method
The male Kunming mice (25-30g) were randomly divided into the sham operation group, the SAH group, the Tregs deletion group and the PBS injection group. The subarachnoid hemorrhage model was established by intravascular puncture, and the change of the blood flow of the parietal cortex was recorded by the laser Doppler haemorrhage. The model of the non perforated.Tregs deletion group in the sham operation group must pass through the mouse abdominal cavity. 0.20mgCD25+ specific antibody (PC61mAb) was injected to construct.PBS intraperitoneal injection group of Treg cell deletion mice and 0.20mg phosphate buffer solution was injected intraperitoneally. After the mice were killed, the brain was quickly taken and the hemorrhage in the basal pool of the brain was photographed with a camera. According to the reports of Sugawara and so on, the bleeding grade was evaluated and the grade of hemorrhage was determined by Garci. Garci The a scoring system scored a score of 18 points. After the subarachnoid hemorrhage 72h, the moisture content of the brain tissue was detected by the dry wet weight comparison method. The blood brain barrier permeability was evaluated by Evans blue. After the operation, the paraffin section was made from the brain of 72h, and the basilar artery tube was observed under the microscope. Changes in diameter and wall thickness. After making brain sections, Nissl staining was made to observe the changes of nerve cells. The apoptosis of brain cells was observed by TUNEL staining. The expression and distribution of IL6 and GFAP in the cortex were measured by immunofluorescence. The content of IL-6, TNF- alpha in brain tissue was measured by enzyme linked immunosorbent assay. The inflammatory factors of cortical tissue were detected by Western blot. The content of IL-6.
Research results
1. the degree of bleeding: there was no bleeding in the sham operation group and no bleeding in the sham operation group; the subarachnoid hemorrhage group, the PBS injection group and the Tregs deletion group met the blood in the subarachnoid space, and there was no statistical difference in the amount of bleeding among the groups.
2. nerve function score: the sham operation group score was 18 points, SAH group, PBS injection group compared with the sham group, there was statistical significance (P0.05), Tregs deletion group compared with the PBS injection group, there was statistical significance (P0.05), and the SAH group was also statistically significant (P0.05).
3. the permeability of blood brain barrier and the determination of brain edema: the permeability of blood brain barrier increased and the water content of brain increased in mice after SAH. Compared with group SAH, the permeability of blood brain barrier increased significantly (P0.05) and the water content of brain tissue was significantly increased (P0.05).
4. basilar artery morphology: the basilar artery was obviously spasmodic after subarachnoid hemorrhage in mice, and spasm was more significant after Tregs deletion (P0.05).
5. Nissl staining: after subarachnoid hemorrhage, the number of Nissl bodies in the cortex of mice decreased significantly, and the decrease after Tregs deletion was more significant (P0.05).
6. cell apoptosis detection: after subarachnoid hemorrhage, the number of apoptotic cells in the cortex of mice increased. After the deletion of Tregs, the number of apoptotic cells was significant and significant (P0.05).
7. the immunofluorescence method was used to determine the cortical area IL-6, the GFAP SAH group, the PBS injection group and the Treg cell deletion group increased, and the Tregs deletion group increased more significantly (P0.05).
The content of IL-6 and TNF- alpha in brain tissue was measured by 8.ELISA. The expression of IL-6 and TNF- in the brain tissues of the mice after SAH increased, and the Tregs deletion group was more significant than that in the Tregs deletion group (P0.05).
The expression of IL-6 in IL-6:SAH group, PBS group and Tregs deletion group increased in IL-6:SAH group, PBS group and Tregs deletion group. The Tregs deletion group had significant statistical significance compared with that of the group SAH and the sham group (P0.05).
research conclusion
1. this study successfully constructed a mouse model of subarachnoid hemorrhage and a Tregs deletion model, and found that the deletion of Tregs would aggravate cerebral vasospasm, brain edema, blood brain barrier permeability, apoptosis and so on, and also aggravate the inflammatory injury of brain tissue.
2., after SAH, the nerve function of mice was damaged. After Tregs deletion, the movement and sensory function of mice could be aggravated.

【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

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