本文選題：實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎 + 多西環(huán)素��； 參考：《四川醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:1.觀察多西環(huán)素(DOX)在實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎(EAE)大鼠發(fā)病過程中的防治作用;2.探討DOX對(duì)防治EAE大鼠發(fā)病可能的免疫學(xué)機(jī)制。方法:隨機(jī)將50只雌性Wistar大鼠分為正常對(duì)照組、EAE對(duì)照組、大劑量DOX防治組、中劑量DOX防治組、小劑量DOX防治組5組,每組10只。采用粗制髓鞘堿性蛋白(MBP)和含卡介苗(BCG)的完全福氏佐劑(CFA)制成免疫抗原,皮下注入EAE對(duì)照組及大、中、小劑量DOX防治組大鼠后足掌,同法給正常對(duì)照組大鼠注射等量生理鹽水(NS)。自造模前三天起,分別給予大、中、小劑量DOX防治組大鼠DOX20mg/kg/d、10mg/kg/d、5mg/kg/d腹腔注射,正常對(duì)照組及EAE對(duì)照組大鼠予以等量NS腹腔注射,均連續(xù)10天。將發(fā)病大鼠連續(xù)3天神經(jīng)功能障礙評(píng)分無(wú)加重、四肢癱瘓或死亡時(shí)作為發(fā)病高峰期。觀察大鼠發(fā)病情況,并記錄發(fā)病潛伏期和進(jìn)展期以及高峰期神經(jīng)功能障礙評(píng)分。在大鼠發(fā)病高峰期終止實(shí)驗(yàn),處死大鼠,未發(fā)病大鼠及正常對(duì)照組大鼠4周后處死,并留取眶靜脈血、腦脊液(CSF)、腦脊髓組織、脾臟組織。將腦脊髓組織行HE染色并在光鏡下觀察其病理學(xué)改變及程度;用酶聯(lián)免疫吸附(ELISA)法檢測(cè)大鼠發(fā)病高峰期外周血單個(gè)核細(xì)胞(PBMC)分泌IL-4、IL-17、IL-23、IFN-γ、TGF-β1水平及腦組織勻漿MMP-2、MMP-9水平;流式細(xì)胞儀測(cè)定脾臟Th17、CD4+CD25+Foxp3+Treg細(xì)胞亞群分布比率;測(cè)定血清和CSF白蛋白含量,計(jì)算CSF和血清白蛋白含量比值即QA值,評(píng)估血腦屏障(BBB)功能。所獲得的數(shù)據(jù)通過SPSS19.0軟件包進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:(1)大鼠發(fā)病情況:正常對(duì)照組大鼠未見異常。eae對(duì)照組及大、中、小劑量dox防治組發(fā)病率分別為90%、40%、60%、80%。eae對(duì)照組及大、中、小劑量dox防治組發(fā)病潛伏期分別為11.3±1.5天、23.7±1.9天、20.6±1.2天、17.7±1.0天,各劑量dox防治組發(fā)病潛伏期均較eae對(duì)照組延長(zhǎng)(p0.01),大劑量dox防治組大鼠發(fā)病潛伏期延長(zhǎng)最明顯,各劑量dox防治組間差異有統(tǒng)計(jì)學(xué)意義(p0.01)。eae對(duì)照組及大、中、小劑量dox防治組發(fā)病進(jìn)展期分別為10.1±1.0天、4.4±0.6天、6.9±0.4天、7.7±0.7天,各劑量dox防治組發(fā)病進(jìn)展期均較eae對(duì)照組縮短(p0.01),其中以大劑量組大鼠發(fā)病進(jìn)展期縮短最明顯,各劑量dox防治組間差異有統(tǒng)計(jì)學(xué)意義(p0.01或p0.05)。eae對(duì)照組及大、中、小劑量dox防治組大鼠發(fā)病高峰期神經(jīng)功能障礙評(píng)分分別為4.3±0.7分、1.1±0.4分、1.9±0.3分、3.0±0.3分,各劑量dox防治組神經(jīng)功能障礙評(píng)分均較eae對(duì)照組降低(p0.01),其中以大劑量組降低最明顯,各劑量dox防治組間差異有統(tǒng)計(jì)學(xué)意義(p0.01)。(2)病理學(xué)改變:正常對(duì)照組大鼠腦脊髓組織未見明顯異常。eae對(duì)照組大鼠腦脊髓組織內(nèi)可見小血管周圍以淋巴細(xì)胞為主的大量炎性細(xì)胞浸潤(rùn),并伴少許單核細(xì)胞,典型者形成“血管套”樣改變。而各劑量dox防治組病理改變均較eae對(duì)照組減輕,其中以大劑量dox防治組最輕。(3)dox對(duì)eae大鼠發(fā)病高峰期pbmc分泌ifn-γ、il-4水平及ifn-γ/il-4比值的影響:eae對(duì)照組和各劑量dox防治組大鼠發(fā)病高峰期pbmc分泌ifn-γ水平、ifn-γ/il-4比值較正常對(duì)照組升高,eae對(duì)照組和中小劑量dox防治組大鼠發(fā)病高峰期pbmc分泌il-4水平較正常組降低(p0.01),大劑量組ifn-γ水平較正常對(duì)照組改變不明顯(p0.05);各劑量dox防治組大鼠pbmc分泌ifn-γ水平、ifn-γ/il-4比值較eae對(duì)照組降低、il-4水平較eae對(duì)照組升高(p0.01);隨dox劑量增加,各劑量dox防治組大鼠pbmc分泌ifn-γ水平及ifn-γ/il-4比值越低、il-4水平越高,除大劑量dox防治組大鼠pbmc分泌il-4水平較中劑量組改變不明顯外(p0.05),余各組間差異有統(tǒng)計(jì)學(xué)意義(p0.01)。(4)dox對(duì)eae大鼠發(fā)病高峰期pbmc分泌il-17、il-23、tgf-β1水平的影響:除大劑量dox防治組pbmc分泌il-17、il-23水平較正常對(duì)照組升高不明顯(p0.05)外,eae對(duì)照組和各劑量dox防治組大鼠發(fā)病高峰期pbmc分泌il-17、il-23水平較正常對(duì)照組升高,tgf-β1水平較正常對(duì)照組降低(p0.01);各劑量dox防治組大鼠pbmc分泌il-17、il-23水平較eae對(duì)照組降低、tgf-β1水平較eae對(duì)照組升高(p0.01);隨dox劑量增加,各劑量dox防治組大鼠pbmc分泌il-17、il-23水平越低、tgf-β1水平越高,除大劑量dox防治組大鼠pbmc分泌tgf-β1水平較中劑量組改變不明顯外(p0.05),余各組間差異有統(tǒng)計(jì)學(xué)意義(p0.01)。(5)dox對(duì)eae大鼠發(fā)病高峰期脾臟th17、cd4+cd25+foxp3+treg細(xì)胞亞群分布比率的影響:eae對(duì)照組和中、小劑量dox防治組大鼠發(fā)病高峰期脾臟th17細(xì)胞比率較正常對(duì)照組升高,脾臟cd4+cd25+foxp3+treg細(xì)胞比率較正常對(duì)照組降低(p0.01),大劑量dox防治組大鼠發(fā)病高峰期脾臟th17細(xì)胞、cd4+cd25+foxp3+treg細(xì)胞比率較正常組改變不明顯(p0.05);各劑量dox防治組大鼠脾臟th17細(xì)胞比率較eae對(duì)照組降低、脾臟cd4+cd25+foxp3+treg細(xì)胞比率較eae對(duì)照組升高(p0.01);隨dox劑量增加,各劑量dox防治組大鼠脾臟th17細(xì)胞比率越低、cd4+cd25+foxp3+treg細(xì)胞比率越高,各劑量dox防治組間差異有統(tǒng)計(jì)學(xué)意義。(6)dox對(duì)eae大鼠發(fā)病高峰期腦組織mmp-2、mmp-9水平及qa值的影響:除大劑量dox防治組大鼠腦組織mmp-2水平較正常對(duì)照組升高不明顯(p0.05)外,eae對(duì)照組及各劑量dox防治組大鼠發(fā)病高峰期腦組織mmp-2、mmp-9水平及qa值較正常對(duì)照組明顯升高(p0.01或p0.05),各劑量dox防治組大鼠發(fā)病高峰期腦組織mmp-2、mmp-9水平及qa值較eae對(duì)照組明顯降低(p0.01),隨dox劑量增加,各劑量dox防治組大鼠腦組織mmp-2、mmp-9水平及qa值水平越低,各dox防治組間差異有統(tǒng)計(jì)學(xué)意義(p0.01或p0.05)。(7)相關(guān)性分析:eae對(duì)照組及各劑量dox防治組大鼠發(fā)病潛伏期與高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17細(xì)胞比率呈負(fù)相關(guān)(p0.01或p0.05),與發(fā)病高峰期tgf-β1、cd4+cd25+foxp3+treg細(xì)胞比率呈正相關(guān);eae對(duì)照組及各劑量dox防治組大鼠發(fā)病進(jìn)展期與高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17細(xì)胞比率呈正相關(guān)(p0.01或p0.05),與發(fā)病高峰期tgf-β1、cd4+cd25+foxp3+treg細(xì)胞比率呈負(fù)相關(guān);eae對(duì)照組及各劑量dox防治組大鼠發(fā)病高峰期神經(jīng)功能障礙評(píng)分與高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17細(xì)胞比率呈正相關(guān)(p0.01或p0.05),與發(fā)病高峰期tgf-β1、cd4+cd25+foxp3+treg細(xì)胞比率呈負(fù)相關(guān)。結(jié)論:1.在eae發(fā)病高峰期,大鼠外周血促炎因子增多、抑炎因子減少,脾臟th17比率增高、cd4+cd25+foxp3+treg細(xì)胞比率降低,存在體內(nèi)th1/th2平衡及th17/cd4+cd25+foxp3+treg平衡漂移。2.在eae發(fā)病高峰期,大鼠腦組織MMP-2、MMP-9水平升高以及腦脊液和血清蛋白比值QA值升高,提示BBB破壞。3.DOX可降低EAE大鼠的發(fā)病率,延長(zhǎng)EAE大鼠發(fā)病潛伏期、縮短進(jìn)展期、降低發(fā)病高峰期神經(jīng)功能障礙評(píng)分,減輕CNS炎癥細(xì)胞浸潤(rùn),提示DOX對(duì)EAE發(fā)病有防治作用。4.DOX通過抑制促炎性細(xì)胞因子的生成、促進(jìn)抑炎性細(xì)胞因子的釋放,降低脾臟Th17比率、升高CD4+CD25+Foxp3+Treg細(xì)胞比率,降低腦組織MMP-2、MMP-9水平及QA值,糾正Th1/Th2、Th17/CD4+CD25+Foxp3+Treg平衡失調(diào),保護(hù)BBB,從而對(duì)EAE的發(fā)病起防治作用。
[Abstract]:Objective: 1. to observe the preventive and therapeutic effects of doxycycline (DOX) in the pathogenesis of experimental allergic encephalomyelitis (EAE) rats; 2. to explore the possible immunological mechanism of DOX on the prevention and treatment of EAE rats. Methods: 50 female Wistar rats were randomly divided into normal control group, EAE control group, large dose DOX control group, medium dose DOX control group, small dose of EAE. The 5 groups of DOX control group, 10 rats in each group, were treated with crude myelin basic protein (MBP) and complete FOS adjuvant (CFA) containing Calmette Guerin (BCG) to make immune antigen, subcutaneous injection of EAE control group and large, medium and small dose DOX control group to the hind foot palms of rats, and the same method was given to normal control group with equal amount of normal saline (NS). From the first three days before the self-made model, they were given respectively. Rats in the large, medium and small dose DOX control group were intraperitoneally injected with DOX20mg/kg/d, 10mg/kg/d, 5mg/kg/d, the normal control group and the EAE control group were given the same amount of NS intraperitoneal injection for 10 days. The scores of the neurological dysfunction were not aggravated, the extremities paralyzed or died as the peak period. The incidence of the rats was observed and the hair was recorded and recorded. The latency and progression of the disease and the peak nerve dysfunction score. The rats were killed at the peak period of the onset of the disease. The rats were killed, the rats in the uninfected rats and the normal control rats were killed 4 weeks later, and the orbital vein blood, cerebrospinal fluid (CSF), the brain spinal tissue and the spleen tissue were retained. The brain and spinal tissue were stained with HE and the pathological changes were observed under the light microscope. Change and degree; detect the secretion of IL-4, IL-17, IL-23, IFN- gamma, TGF- beta 1 and MMP-2, MMP-9 level of the brain homogenate by enzyme linked immunosorbent assay (ELISA) method, and the ratio of Th17, CD4+CD25+Foxp3+Treg cell subgroup distribution in the spleen, and the determination of the content of serum and CSF albumin by flow cytometry. The blood brain barrier (BBB) function was evaluated by the ratio of serum albumin to serum albumin (QA). The data obtained by the SPSS19.0 software package were statistically analyzed. Results: (1) the incidence of the rats in the normal control group was not found in the abnormal.Eae control group and in the large, medium, and small dose DOX control group, respectively, and the incidence of the control group was 90%, 40%, and 80%.eae control group and large, medium, small, small, small, small, and small. The incubation period of the dose DOX control group was 11.3 + 1.5 days, 23.7 + 1.9 days, 20.6 + 1.2 days, 17.7 + 1 days. The onset latency of each dose DOX control group was longer than that of the EAE control group (P0.01), and the onset latency of the large dose DOX control group was the most obvious, and the difference between each dose of DOX control group was statistically significant (P0.01).Eae control group and large, medium, The progression of small dose DOX control group was 10.1 + 1 days, 4.4 + 0.6 days, 6.9 + 0.4 days, 7.7 + 0.7 days, and the progression of DOX control group was shorter than that of EAE control group (P0.01), which was most obvious in the large dose group, and the difference between the dox control group was statistically significant (P0.01 or P0.05).Eae control group. The scores of the high, medium and small dose DOX control group were 4.3 + 0.7, 1.1 + 0.4, 1.9 + 0.3 and 3 + 0.3 respectively. The scores of nerve dysfunction in each dose DOX control group were lower than those of the EAE control group (P0.01), which was the most obvious in the large dose group, and the difference between the DOX control groups was statistically significant (P0.01 (2) pathological changes: in the normal control group, the brain and spinal cord of the normal control group had no obvious abnormality in the.Eae control group. The infiltration of large amounts of inflammatory cells around the small vessels in the brain and spinal cord of the rats was observed, and a few mononuclear cells were accompanied by a few mononuclear cells. The pathological changes of the DOX control group were compared with that of the EAE control group. Group DOX was lightest. (3) the effect of DOX on the secretion of ifn- gamma, IL-4 level and the ratio of ifn- gamma /il-4 at the peak period of the onset of the onset of EAE rats: EAE control group and DOX control group of each dose of DOX control group, PBMC secretion of ifn- gamma level, increased ratio of gamma ray to normal control group, control group and small and medium dose control group. The IL-4 level of PBMC secreted at the peak period of the rats was lower than that in the normal group (P0.01), and the level of ifn- gamma in the large dose group was not significantly higher than that in the normal control group (P0.05), and the PBMC secretion of ifn- gamma in the DOX control group was lower than that in the EAE control group, and the IL-4 level was higher than that in the control group. The lower the level of ifn- gamma and the ratio of ifn- gamma /il-4 to PBMC, the higher the IL-4 level, the higher the level of IL-4 in the DOX control group except the middle dose group (P0.05), the difference between the remaining groups was statistically significant (P0.01). (4) DOX on the high peak period of the pathogenesis of EAE rats: except for the big dose The level of PBMC secreted IL-17 in DOX control group, IL-23 level was not significantly higher than that in normal control group (P0.05), EAE control group and DOX control group of EAE control group, PBMC secreted IL-17, IL-23 level was higher than normal control group, tgf- beta 1 level was lower than normal control group (P0.01). The level of tgf- beta 1 in the e control group was higher than that in the EAE control group (P0.01). With the increase of DOX dose, the PBMC secreted IL-17 in the DOX control group, the lower the IL-23 level, the higher the tgf- beta 1 level, the greater the level of PBMC secreted beta 1 than the middle dose group. (5). ) the effect of DOX on the ratio of Th17 and cd4+cd25+foxp3+treg cell subsets in the spleen of EAE rats: the ratio of spleen Th17 cells in the EAE control group and the small dose DOX control group was higher than that in the normal control group, and the ratio of cd4+cd25+foxp3+treg cells in the spleen was lower than that in the normal control group (P0.01), and the large dose of DOX control group of the rats was in the control group. The ratio of cd4+cd25+foxp3+treg cells in the spleen Th17 cells was not significantly higher than that in the normal group (P0.05), and the ratio of spleen Th17 cells in the control group of DOX control group was lower than that of the EAE control group, and the proportion of cd4+cd25+foxp3+treg cells in the spleen was higher than that of the EAE control group (P0.01). With the increase of DOX dose, the spleen Th17 cells of the rats of the DOX control group were all dosed. The lower the ratio, the higher the ratio of cd4+cd25+foxp3+treg cells, the difference between the different doses of DOX in the control group was statistically significant. (6) the effect of DOX on the MMP-2, MMP-9 level and QA value of the brain tissue at the peak of the onset of EAE rats: the level of MMP-2 in the brain tissue of the rats of the large dose DOX control group was not significantly higher than that in the normal control group (P0.05), the EAE control group and the dose DOX. The brain tissue MMP-2, MMP-9 level and QA value in the control group were significantly higher than those of the normal control group (P0.01 or P0.05). The level of MMP-2, MMP-9 level and QA in each dose of DOX control group were significantly lower than those of the EAE control group (P0.01). The lower the value level, the difference between the DOX control groups was statistically significant (P0.01 or P0.05). (7) correlation analysis: the ratio of the latency period and the peak period ifn- gamma /il-4 in the EAE control group and the DOX control group of each dose group, IL-17, MMP-2, MMP-9, QA, the Th17 cell ratio was negatively correlated with the peak period of the onset of the disease. The ratio of EAE control group and DOX control group was positively correlated with the ratio of ifn- gamma /il-4 at peak period, IL-17, MMP-2, MMP-9, QA, Th17 cell ratio (P0.01 or P0.05), which was negatively correlated with the ratio of tgf- beta 1 at the peak stage of onset, and the peak of the incidence of rats in the control group and each dose control group. The ratio of ifn- gamma /il-4, IL-17, MMP-2, MMP-9, QA value, Th17 cell ratio was positively correlated with the ratio of Th17 cells (P0.01 or P0.05), and was negatively correlated with the ratio of tgf- beta 1 and cd4+cd25+foxp3+treg cells at the peak period. Conclusion: 1. in the peak of the onset of EAE, the increase of proinflammatory factors in the peripheral blood, the decrease of anti inflammatory factors and the increase of spleen ratio were increased. The ratio of cd4+cd25+foxp3+treg cells decreased, the balance of th1/th2 in the body and the drift of th17/cd4+cd25+foxp3+treg balance.2. at the peak of EAE, the increase of MMP-2 and MMP-9 in the brain tissue and the increase of the ratio of cerebrospinal fluid and serum protein, suggesting that BBB destruction of.3.DOX could reduce the incidence of EAE rats and prolong the incubation period of the EAE rats. To shorten the stage of progression, reduce the score of neurological dysfunction at the peak of onset and reduce the infiltration of CNS inflammatory cells, suggesting that DOX has a preventive effect on the pathogenesis of EAE,.4.DOX can promote the release of anti inflammatory cytokines by inhibiting the formation of proinflammatory cytokines, reduce the Th17 ratio of the spleen, increase the ratio of CD4+CD25+Foxp3+Treg cells, and reduce the MMP-2, MMP- of the brain tissue. 9 level and QA value, correct Th1/Th2, Th17/CD4+CD25+Foxp3+Treg imbalance, protect BBB, thereby preventing and controlling the onset of EAE.
【學(xué)位授予單位】：四川醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R744.5

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相關(guān)期刊論文 前3條

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