【摘要】：目的：蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)是常見的腦組織出血性病變,因其致死致殘率高,且存活者多預后不良,造成患者生活質(zhì)量下降。研究證實SAH患者腦血管痙攣(cerebralvasospasm, CVS)和早期腦損傷(Early brain injury, EBI)的嚴重程度,與不良預后相關(guān)。因此,非常有必要研制新型藥物,控制SAH后早期炎性免疫反應,減輕EBI和CVS的致病性,減輕患者的痛苦。有研究證實,調(diào)節(jié)性T細胞(CD4+CD25+regulatory T cell, Treg)在抑制腦梗死再灌注小鼠腦組織的損傷方面發(fā)揮內(nèi)源性保護作用。那么,同樣做為顱內(nèi)缺血缺氧性損傷,調(diào)節(jié)性T細胞能否在SAH后的炎性免疫反應中發(fā)揮調(diào)節(jié)作用呢?我們利用線栓刺破法制作小鼠SAH模型,從小鼠脾、淋巴結(jié)中提取并體外擴增調(diào)節(jié)性T細胞,輸入SAH小鼠體內(nèi),通過小鼠神經(jīng)功能學的評價和各項實驗數(shù)據(jù)的分析,探討Treg對SAH小鼠腦保護作用的療效。為驗證體外細胞實驗得到的結(jié)論,能否在動物模型中復制,并進一步探討Treg對體外BV2及體內(nèi)腦組織保護作用的機制和作用方式,我們通過脂多糖激活BV2活化的體外細胞模型,模擬體內(nèi)小鼠腦組織SAH缺血缺氧的病理過程,從小鼠脾臟及淋巴結(jié)中提取并體外擴增調(diào)節(jié)性T細胞,與脂多糖激活的BV2共培養(yǎng),旨在通過Treg與BV2共培養(yǎng),驗證Treg對體外BV2的保護作用機制。我們分別從體外細胞培養(yǎng)和動物在體實驗兩個角度,從細胞整體水平、蛋白水平和mRNA三個水平共同研究,多方位多角度綜合探討Treg發(fā)揮療效的機制。方法：通過免疫磁珠雙選的方法,提取小鼠脾臟及淋巴結(jié)Treg。頸內(nèi)動脈線栓刺破法制作小鼠SAH模型,隨機分為Sham組、SAH+PBS組、SAH+SP(Splenocyte,脾細胞)組、SAH+Treg組,經(jīng)股靜脈輸注CD4+CD25+Treg,造模成功后48h,記錄腦血流量及各組動物行為學指標；分別制備石蠟、冰凍切片,通過不同染色法研究腦細胞形態(tài)及陽性細胞的分布情況,驗證Treg輸注移植對SAH小鼠腦組織的保護作用。脂多糖誘導體外BV2激活,Treg與激活的BV2共培養(yǎng),采用MTT法檢測體外BV2的活性；利用硝酸還原酶法和ELISA法檢測炎性因子TNF-α、IL-6、NO、IL-10的含量；采用尼羅紅微球吞噬實驗,驗證Treg對LPS誘導BV2吞噬情況的調(diào)節(jié)作用。免疫熒光雙染及免疫熒光三染法,標記體外BV2和體內(nèi)SAH小鼠腦組織不同極化狀態(tài)M1、M2期陽性細胞表達的情況；采用實時熒光定量PCR (RT-PCR)法分別檢測體外BV2和SAH小鼠腦組織M 1、M2期標志物mRNA水平的表達量；采用Western blot法分別檢測體外小膠質(zhì)細胞和SAH小鼠腦組織內(nèi)相關(guān)信號通道TLR4/p-NFκB、 p-P38/P-ERK1/2的蛋白表達量。通過多方位多角度的綜合研究,深入探討Treg對BV2和SAH小鼠腦組織保護作用的機制。結(jié)果：Treg靜脈輸注移植,SAH后小鼠死亡率下降,各項生命體征及神經(jīng)功能學表現(xiàn)情況明顯好轉(zhuǎn),神經(jīng)功能學評分提高,腦組織血流量改善,小鼠腦水腫情況減輕,腦實質(zhì)內(nèi)微動脈及基底動脈痙攣程度減輕,血管內(nèi)皮細胞受損程度減輕,小鼠腦組織神經(jīng)元凋亡數(shù)量減少,損傷程度降低。Treg與LPS激活的體外BV2共培養(yǎng),炎性刺激因子含量明顯減少,抑制因子IL-10含量增加,同時,BV2在吞噬過程中吞掉了更多的微球,Treg對LPS激活的BV2有明顯保護作用。免疫熒光染色顯示,Treg輸注移植,體外小膠質(zhì)細胞和SAH小鼠腦組織M1期標記物表達減少,M2期標記物表達上調(diào)。RT-PCR結(jié)果顯示,Treg輸注移植,體外小膠質(zhì)細胞和SAH小鼠腦組織M 1期標記物mRNA水平的表達減少,M2期標志物mRNA水平的表達上調(diào)。Western blot結(jié)果顯示,Treg輸注移植,體外BV2和SAH小鼠腦組織內(nèi)炎性信號通路TLR4、p-NF-κB、p-P38、p-ERK1/勺蛋白表達量明顯減少。結(jié)論：1、Treg輸注移植能夠減輕SAH小鼠早期死亡率,改善48h局部腦血流量,有效緩解SAH后血腦屏障的破壞程度,維護腦實質(zhì)內(nèi)血管形態(tài)的完整性,減輕BA痙攣程度,顯著減輕小鼠皮層及海馬神經(jīng)元損傷的程度,減輕腦組織細胞過度凋亡的程度,有助于改善SAH后小鼠神經(jīng)功能學水平,促進神經(jīng)元的修復,提高SAH后小鼠生活質(zhì)量。2、Treg與LPS激活的BV2共培養(yǎng),細胞培養(yǎng)液炎性刺激因子的含量減少,抑制因子的含量增加,BV2的吞噬作用增強。3、Treg輸注治療可通過減少M1極化狀態(tài)標記物mRNA水平的表達,抑制小膠質(zhì)細胞M1極化；增加M2極化狀態(tài)標記物mRNA水平的表達,促進小膠質(zhì)細胞M2極化,從而抑制炎性細胞因子的分泌,發(fā)揮腦組織的保護功效。4、Treg可能通過抑制炎性信號通路TLR4/p-NF-κB、p-P38/p-ERK1/2的激活,減少炎性刺激因子的表達。本實驗創(chuàng)新性地將RT-PCR法和免疫熒光染色法相結(jié)合,同時研究Treg對體外BV2和動物體內(nèi)小膠質(zhì)細胞不同極化狀態(tài)的調(diào)節(jié)作用,通過體外細胞水平的實驗與在體動物模型的實驗相結(jié)合,將動物的行為學觀察與細胞分子水平的機制研究相結(jié)合,多方面多角度充分地探討了調(diào)節(jié)性T細胞對組織。的保護療效。本實驗證明,Treg對體外BV2和SAH小鼠腦組織有明顯的保護作用。然而,動物模型水平得出的結(jié)論,與臨床人體的實際應用仍有較大距離,細胞作為一種生物學活性物質(zhì),不能像化學藥物一樣穩(wěn)定,其保存及運輸?shù)确矫娲嬖诘膯栴}是臨床應用的重要瓶頸。另外,人體活體細胞的移植,尚存在倫理及排異等多方面的問題有待進一步研究和解決。
[Abstract]:Objective: The subarachnoid hemorrhage (SAH) is a common hemorrhagic lesion of the brain, because of its high death rate and the poor prognosis of the survivors, the quality of life of the patient is decreased. The severity of cerebral vasospasm (CVS) and early brain injury (EBI) in SAH patients was confirmed to be related to the poor prognosis. Therefore, it is necessary to develop new drugs, to control the early inflammatory immune response after SAH, to reduce the pathogenicity of EBI and CVS, and to reduce the pain of patients. It has been found that regulatory T cells (CD4 + CD25 + regulatory T cell, Treg) play an endogenous protective role in the inhibition of brain tissue damage in the brain of cerebral infarction. So, can regulatory T-cells play a regulatory role in the inflammatory immune response after SAH? In order to study the effect of Treg on the brain protection of SAH mice, we made the SAH model of the mouse by means of the line-bolt puncture method, and extracted and expanded the regulatory T-cells in vitro from the spleen and the lymph nodes of the mouse, and the regulatory T cells were amplified in vitro. The effect of Treg on the brain protection of the SAH mice was discussed by the evaluation of the neurological function and the analysis of the experimental data. In order to verify the conclusion of in vitro cell experiment, it is possible to replicate in animal model, and to further study the mechanism and function of Treg on the in vitro BV2 and in vivo brain tissue protection. We activate BV2-activated in vitro cell model by lipopolysaccharides. In order to study the pathological process of SAH ischemia and hypoxia in the mouse brain, the regulatory T cells were extracted from the spleen and lymph nodes of the mouse and the regulatory T cells were expanded in vitro, co-cultured with the BV2-activated BV2, and the mechanism of the protective effect of Treg on the in vitro BV2 was verified by co-culture of Treg and BV2. From the two angles of in vitro cell culture and in vivo experiment, we studied the mechanism of Treg's therapeutic effect from the whole level, the level of protein and the three levels of mRNA. Methods: The spleen of the mouse and the lymph node Treg were extracted by double-selection of the immunomagnetic beads. The SAH model of the mouse was made by the internal carotid artery bolt puncture method. The SAH model was randomly divided into the sham group, the SAH + PBS group, the SAH + SP (Splasmote, the spleen cell) group, the SAH + Treg group, the transfemoral vein infusion of CD4 + CD25 + Treg, the cerebral blood flow of the brain and the behavioral indexes of the animals in each group. The protective effect of Treg infusion on the brain tissue of SAH mice was verified by the study of the distribution of the morphology of the brain cells and the positive cells in different staining methods. Lipopolysaccharide induced in vitro BV2 activation, Treg was co-cultured with activated BV2, and the activity of the in vitro BV2 was detected by MTT method; the content of TNF-1, IL-6, NO, IL-10 in the inflammatory factors was detected by the nitric acid reductase method and the ELISA method; To verify the effect of Treg on the phagocytosis of BV2 induced by LPS. Immunofluorescence double staining and immunofluorescence three-staining method were used to mark the expression of M1 and M2 positive cells in different polarization states of the brain tissue of in vitro BV2 and in vivo SAH mice, and the in vitro BV2 and SAH mouse brain tissue M 1 were detected by real-time fluorescence quantitative PCR (RT-PCR). The expression of marker mRNA in M2 phase was detected by Western blot. The expression of TLR4/ p-NF-B, p-P38/ P-ERK1/2 was detected by Western blot. The mechanism of the protective effect of Treg on the brain tissue of BV2 and SAH mice was discussed through a comprehensive study of multi-angle and multi-angle. Results: After the infusion of Treg vein, the mortality of the mice after SAH decreased, the performance of various vital signs and the neurological function improved, the score of the neurological function was improved, the blood flow of the brain tissue was improved, the cerebral edema of the mice was relieved, and the degree of cerebral parenchymal micro-artery and basilar artery spasm was relieved. The degree of damage to the vascular endothelial cells was reduced, and the number of neuronal apoptosis in the brain of the mice was reduced and the degree of damage was reduced. Treg was co-cultured with LPS-activated in vitro BV2, the content of inflammatory stimulating factor was significantly reduced, and the content of IL-10 was increased. At the same time, BV2 swallowed more microspheres during the phagocytosis, and Treg had a significant protective effect on LPS-activated BV2. Immunofluorescence staining showed a decrease in the expression of the marker in the M1 phase of the brain tissue of the in vitro microglia and SAH mice, and the expression of the marker in the M2 phase was up-regulated. The results of RT-PCR showed that the expression of the mRNA level of the marker mRNA in the brain tissue of the in vitro and in vitro microglia and SAH mice was decreased, and the expression of the expression of the marker mRNA in the M2 phase was up-regulated. Western blot showed that the expression of the expression of TLR4, p-NF-B, p-P38, p-ERK1/ spoon in the brain tissue of BV2 and SAH mice in vitro was significantly reduced. Conclusion:1, Treg infusion can reduce the early death rate of SAH mice, improve the local cerebral blood flow of 48 h, effectively relieve the degree of destruction of the blood-brain barrier after SAH, maintain the integrity of the blood vessel in the parenchymal vessels, and reduce the degree of BA spasm, the degree of damage to the cortex of the mouse and the neuron of the hippocampus is obviously reduced, the degree of the over-apoptosis of the brain tissue cells is reduced, the level of the neural function of the mouse after the SAH is improved, the repair of the neuron is promoted, the quality of the life of the mouse after SAH is improved, and 2, Treg is co-cultured with the LPS-activated BV2, the content of the inflammatory stimulation factor of the cell culture solution is reduced, the content of the inhibitory factor is increased, the phagocytosis of the BV2 is enhanced, and 3, the Treg infusion therapy can inhibit the polarization of the microglia M1 by reducing the expression of the mRNA level of the M1 polarized state marker, and increase the expression of the mRNA level of the M2 polarized state marker, It is possible to reduce the expression of inflammatory stimulus by inhibiting the activation of the inflammatory signal pathway TLR4/ p-NF-SupB, p-P38/ p-ERK1/2. In this experiment, RT-PCR and immunofluorescence staining were used to study the effects of Treg on the different polarization states of microglia in vitro and in vivo. By combining the behavior of animal and the mechanism of cellular molecular level, the effect of regulatory T cell on the tissue was discussed in various aspects. And the protective effect is improved. The experimental results show that Treg has a significant protective effect on the brain tissue of BV2 and SAH mice in vitro. However, the results of animal model's level show that there is still a great distance from the actual application of the clinical human body. As a kind of biological active substance, the cell can't be as stable as the chemical medicine, and the problems in the preservation and transportation of the cell are the important bottleneck of clinical application. In addition, the transplantation of living cells of human body, there are still many problems, such as ethics and exclusion, to be further studied and solved.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R743.35

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