本文選題：內(nèi)源性調(diào)節(jié)性T細胞 + 蛛網(wǎng)膜下腔出血　； 參考：《泰山醫(yī)學院》2014年碩士論文

【摘要】：研究目的 1.探討內(nèi)源性CD4+CD25+調(diào)節(jié)性T細胞刪除后對蛛網(wǎng)膜下腔出血后小鼠神經(jīng)功能的影響。 2.探討內(nèi)源性調(diào)節(jié)性T細胞刪除對蛛網(wǎng)膜下腔出血后早期腦損傷的影響。 3.通過酶聯(lián)免疫吸附法測定皮層IL-6、 TNF-α,初步探討CD4+CD25+調(diào)節(jié)性T細胞刪除對腦組織炎癥的影響。 4.通過免疫熒光法檢測皮層區(qū)炎癥因子IL-6、GFAP的含量，通過westernblot檢測腦組織中IL-6的含量，進一步探討內(nèi)源性CD4+CD25+調(diào)節(jié)性T細胞刪除對蛛網(wǎng)膜下腔出血后腦組織炎癥的影響。 研究方法 將雄性昆明小鼠（25-30g），隨機分入假手術組、SAH組、Tregs刪除組以及PBS注射組中。用血管內(nèi)穿刺法建立小鼠蛛網(wǎng)膜下腔出血模型，激光多普勒血流儀記錄頂葉皮層腦組織血流量的變化。假手術組不穿破血管。Tregs刪除組模型需經(jīng)過小鼠腹腔注射0.20mgCD25+特異性抗體（PC61mAb），來構(gòu)建Treg細胞缺失小鼠。PBS腹腔注射組，經(jīng)腹腔注射0.20mg的磷酸鹽緩沖液。術后處死小鼠后,快速取腦,并用照相機拍攝腦基底池部出血情況,根據(jù)Sugawara等的報道,進行出血量評分,判定出血等級。采用Garcia評分系統(tǒng)對運動感覺進行評分,總分為18分。于蛛網(wǎng)膜下腔出血后72h，，用干濕重比較法檢測腦組織含水率。用伊文思藍法評價各組動物血腦屏障通透性。于術后72h，取腦制成石蠟切片，行蘇木素-伊紅染色，于鏡下觀察各組小鼠基底動脈管徑大小以及管壁厚度的變化。制作腦切片后尼氏染色觀察神經(jīng)細胞變化。用TUNEL染色觀察腦部細胞凋亡。用免疫熒光法測定皮層IL6、GFAP的表達及分布情況。用酶聯(lián)免疫吸附法測定腦組織中IL-6、TNF-α含量。用western blot檢測皮層組織炎癥因子IL-6的含量。 研究結(jié)果 1.出血程度：假手術組無出血，假手術組無出血；蛛網(wǎng)膜下腔出血組、PBS注射組、Tregs刪除組在蛛網(wǎng)膜下腔均見到血液，各組之間出血量無統(tǒng)計意義的差異。 2.神經(jīng)功能評分：假手術組評分為18分， SAH組、 PBS注射組與假手術組評分相比，有統(tǒng)計學意義(P0.05)，Tregs刪除組與PBS注射組評分相比，有統(tǒng)計學意義(P0.05)，與SAH組的評分相比也有統(tǒng)計學意義(P0.05)。 3.血腦屏障通透性及腦水腫測定：小鼠SAH后血腦屏障通透性增加，腦組織含水量加重；Tregs刪除組與SAH組相比，血腦屏障通透性顯著增加(P0．05)，腦組織含水量顯著加重(P0.05)。 4.基底動脈形態(tài)：小鼠蛛網(wǎng)膜下腔出血后基底動脈明顯痙攣，Tregs刪除后痙攣更為顯著(P0.05)。 5.尼氏染色：蛛網(wǎng)膜下腔出血后，小鼠皮層中尼氏小體的神經(jīng)元數(shù)量明顯減少，Tregs刪除后減少更為顯著(P0.05)。 6.細胞凋亡檢測：蛛網(wǎng)膜下腔出血后，小鼠皮層區(qū)凋亡細胞數(shù)增多，Tregs刪除后與SAH后相比,有顯著統(tǒng)計學以及意義（P0.05) 7.免疫熒光法測定皮層區(qū)IL-6、 GFAP SAH組、 PBS注射組、 Treg細胞刪除組皮層區(qū)表達量均增多，Tregs刪除組增多更為顯著(P0．05)。 8.ELISA測定腦組織中IL-6和TNF-α含量： SAH后小鼠腦組織中IL-6和TNF-α表達量均有所升高， Tregs刪除組與之相比更顯著(P0.05) 9.Western blot檢測皮層區(qū)IL-6： SAH組、 PBS組、 Tregs刪除組的腦組織中IL-6表達量均增多，Tregs刪除組與SAH組、假手術組相比有顯著統(tǒng)計學意義(P0.05)。 研究結(jié)論 1.本研究成功構(gòu)建了蛛網(wǎng)膜下腔出血小鼠模型以及Tregs刪除小鼠模型，并發(fā)現(xiàn)Tregs刪除后會加重腦血管痙攣、腦組織水腫、血腦屏障通透性、凋亡等，同時也加重腦組織的炎性損傷。 2.小鼠SAH后產(chǎn)生神經(jīng)功能有所損害，Tregs刪除后可加重小鼠運動、感覺功能的損傷。
[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.

【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.3

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