多西环素对实验性变态反应性脑脊髓炎防治作用的研究

发布时间：2018-06-15 16:50

本文选题：实验性变态反应性脑脊髓炎 + 多西环素　；参考：《四川医科大学》2015年硕士论文

【摘要】：目的:1.观察多西环素(DOX)在实验性变态反应性脑脊髓炎(EAE)大鼠发病过程中的防治作用;2.探讨DOX对防治EAE大鼠发病可能的免疫学机制。方法:随机将50只雌性Wistar大鼠分为正常对照组、EAE对照组、大剂量DOX防治组、中剂量DOX防治组、小剂量DOX防治组5组,每组10只。采用粗制髓鞘碱性蛋白(MBP)和含卡介苗(BCG)的完全福氏佐剂(CFA)制成免疫抗原,皮下注入EAE对照组及大、中、小剂量DOX防治组大鼠后足掌,同法给正常对照组大鼠注射等量生理盐水(NS)。自造模前三天起,分别给予大、中、小剂量DOX防治组大鼠DOX20mg/kg/d、10mg/kg/d、5mg/kg/d腹腔注射,正常对照组及EAE对照组大鼠予以等量NS腹腔注射,均连续10天。将发病大鼠连续3天神经功能障碍评分无加重、四肢瘫痪或死亡时作为发病高峰期。观察大鼠发病情况,并记录发病潜伏期和进展期以及高峰期神经功能障碍评分。在大鼠发病高峰期终止实验,处死大鼠,未发病大鼠及正常对照组大鼠4周后处死,并留取眶静脉血、脑脊液(CSF)、脑脊髓组织、脾脏组织。将脑脊髓组织行HE染色并在光镜下观察其病理学改变及程度;用酶联免疫吸附(ELISA)法检测大鼠发病高峰期外周血单个核细胞(PBMC)分泌IL-4、IL-17、IL-23、IFN-γ、TGF-β1水平及脑组织匀浆MMP-2、MMP-9水平;流式细胞仪测定脾脏Th17、CD4+CD25+Foxp3+Treg细胞亚群分布比率;测定血清和CSF白蛋白含量,计算CSF和血清白蛋白含量比值即QA值,评估血脑屏障(BBB)功能。所获得的数据通过SPSS19.0软件包进行统计学分析。结果:(1)大鼠发病情况:正常对照组大鼠未见异常。eae对照组及大、中、小剂量dox防治组发病率分别为90%、40%、60%、80%。eae对照组及大、中、小剂量dox防治组发病潜伏期分别为11.3±1.5天、23.7±1.9天、20.6±1.2天、17.7±1.0天,各剂量dox防治组发病潜伏期均较eae对照组延长(p0.01),大剂量dox防治组大鼠发病潜伏期延长最明显,各剂量dox防治组间差异有统计学意义(p0.01)。eae对照组及大、中、小剂量dox防治组发病进展期分别为10.1±1.0天、4.4±0.6天、6.9±0.4天、7.7±0.7天,各剂量dox防治组发病进展期均较eae对照组缩短(p0.01),其中以大剂量组大鼠发病进展期缩短最明显,各剂量dox防治组间差异有统计学意义(p0.01或p0.05)。eae对照组及大、中、小剂量dox防治组大鼠发病高峰期神经功能障碍评分分别为4.3±0.7分、1.1±0.4分、1.9±0.3分、3.0±0.3分,各剂量dox防治组神经功能障碍评分均较eae对照组降低(p0.01),其中以大剂量组降低最明显,各剂量dox防治组间差异有统计学意义(p0.01)。(2)病理学改变:正常对照组大鼠脑脊髓组织未见明显异常。eae对照组大鼠脑脊髓组织内可见小血管周围以淋巴细胞为主的大量炎性细胞浸润,并伴少许单核细胞,典型者形成“血管套”样改变。而各剂量dox防治组病理改变均较eae对照组减轻,其中以大剂量dox防治组最轻。(3)dox对eae大鼠发病高峰期pbmc分泌ifn-γ、il-4水平及ifn-γ/il-4比值的影响:eae对照组和各剂量dox防治组大鼠发病高峰期pbmc分泌ifn-γ水平、ifn-γ/il-4比值较正常对照组升高,eae对照组和中小剂量dox防治组大鼠发病高峰期pbmc分泌il-4水平较正常组降低(p0.01),大剂量组ifn-γ水平较正常对照组改变不明显(p0.05);各剂量dox防治组大鼠pbmc分泌ifn-γ水平、ifn-γ/il-4比值较eae对照组降低、il-4水平较eae对照组升高(p0.01);随dox剂量增加,各剂量dox防治组大鼠pbmc分泌ifn-γ水平及ifn-γ/il-4比值越低、il-4水平越高,除大剂量dox防治组大鼠pbmc分泌il-4水平较中剂量组改变不明显外(p0.05),余各组间差异有统计学意义(p0.01)。(4)dox对eae大鼠发病高峰期pbmc分泌il-17、il-23、tgf-β1水平的影响:除大剂量dox防治组pbmc分泌il-17、il-23水平较正常对照组升高不明显(p0.05)外,eae对照组和各剂量dox防治组大鼠发病高峰期pbmc分泌il-17、il-23水平较正常对照组升高,tgf-β1水平较正常对照组降低(p0.01);各剂量dox防治组大鼠pbmc分泌il-17、il-23水平较eae对照组降低、tgf-β1水平较eae对照组升高(p0.01);随dox剂量增加,各剂量dox防治组大鼠pbmc分泌il-17、il-23水平越低、tgf-β1水平越高,除大剂量dox防治组大鼠pbmc分泌tgf-β1水平较中剂量组改变不明显外(p0.05),余各组间差异有统计学意义(p0.01)。(5)dox对eae大鼠发病高峰期脾脏th17、cd4+cd25+foxp3+treg细胞亚群分布比率的影响:eae对照组和中、小剂量dox防治组大鼠发病高峰期脾脏th17细胞比率较正常对照组升高,脾脏cd4+cd25+foxp3+treg细胞比率较正常对照组降低(p0.01),大剂量dox防治组大鼠发病高峰期脾脏th17细胞、cd4+cd25+foxp3+treg细胞比率较正常组改变不明显(p0.05);各剂量dox防治组大鼠脾脏th17细胞比率较eae对照组降低、脾脏cd4+cd25+foxp3+treg细胞比率较eae对照组升高(p0.01);随dox剂量增加,各剂量dox防治组大鼠脾脏th17细胞比率越低、cd4+cd25+foxp3+treg细胞比率越高,各剂量dox防治组间差异有统计学意义。(6)dox对eae大鼠发病高峰期脑组织mmp-2、mmp-9水平及qa值的影响:除大剂量dox防治组大鼠脑组织mmp-2水平较正常对照组升高不明显(p0.05)外,eae对照组及各剂量dox防治组大鼠发病高峰期脑组织mmp-2、mmp-9水平及qa值较正常对照组明显升高(p0.01或p0.05),各剂量dox防治组大鼠发病高峰期脑组织mmp-2、mmp-9水平及qa值较eae对照组明显降低(p0.01),随dox剂量增加,各剂量dox防治组大鼠脑组织mmp-2、mmp-9水平及qa值水平越低,各dox防治组间差异有统计学意义(p0.01或p0.05)。(7)相关性分析:eae对照组及各剂量dox防治组大鼠发病潜伏期与高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17细胞比率呈负相关(p0.01或p0.05),与发病高峰期tgf-β1、cd4+cd25+foxp3+treg细胞比率呈正相关;eae对照组及各剂量dox防治组大鼠发病进展期与高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17细胞比率呈正相关(p0.01或p0.05),与发病高峰期tgf-β1、cd4+cd25+foxp3+treg细胞比率呈负相关;eae对照组及各剂量dox防治组大鼠发病高峰期神经功能障碍评分与高峰期ifn-γ/il-4比值、il-17、mmp-2、mmp-9、qa值、th17细胞比率呈正相关(p0.01或p0.05),与发病高峰期tgf-β1、cd4+cd25+foxp3+treg细胞比率呈负相关。结论:1.在eae发病高峰期,大鼠外周血促炎因子增多、抑炎因子减少,脾脏th17比率增高、cd4+cd25+foxp3+treg细胞比率降低,存在体内th1/th2平衡及th17/cd4+cd25+foxp3+treg平衡漂移。2.在eae发病高峰期,大鼠脑组织MMP-2、MMP-9水平升高以及脑脊液和血清蛋白比值QA值升高,提示BBB破坏。3.DOX可降低EAE大鼠的发病率,延长EAE大鼠发病潜伏期、缩短进展期、降低发病高峰期神经功能障碍评分,减轻CNS炎症细胞浸润,提示DOX对EAE发病有防治作用。4.DOX通过抑制促炎性细胞因子的生成、促进抑炎性细胞因子的释放,降低脾脏Th17比率、升高CD4+CD25+Foxp3+Treg细胞比率,降低脑组织MMP-2、MMP-9水平及QA值,纠正Th1/Th2、Th17/CD4+CD25+Foxp3+Treg平衡失调,保护BBB,从而对EAE的发病起防治作用。
[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.
【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R744.5

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