【作者】 张艳丽；

【导师】 马坚妹；

【作者基本信息】 大连医科大学 ， 人体解剖与组织胚胎学， 2016， 博士

【摘要】 研究背景与目的:神经炎症(neuroinflammation)是中枢神经系统(central nervous system,CNS)损伤和疾病的共同病理特征,其病理变化主要包括神经胶质细胞活化,炎性细胞浸润,促炎性细胞因子的表达与释放,血脑屏障通透性增加等。小胶质细胞(microglia,MG)作为CNS内的免疫细胞,是CNS固有免疫的第一道防线,在神经炎症中首先被激活,并在神经炎症的发生和演进中起到重要作用。被激活的MG存在M1和M2两种形态和功能均显著不同的极化类型。M1型又称经典活化(classicalactivation)型,杀灭外来病原体的同时,分泌肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)等促炎性细胞因子,加重炎症和组织损伤。M2型又称为替代活化(alternative activation)型,可清除细胞碎片以及释放大量保护性因子,如IL-10、IL-4和胰岛素样生长因子-I(insulin-like growth factor 1,IGF-I)等,促进损伤组织的重塑和修复。众多研究表明,MG的极化与各种CNS疾病关系密切。不同疾病类型、同一疾病的不同发展阶段,MG表型特征均有明显差别。寻找MG极化原因,进而调控MG极化状态,可能成为CNS疾病全新的治疗措施。神经炎症中MG的活化伴随着多种基因表达的变化,我们之前的研究发现,组织蛋白酶C(cathepsin C,Cat C)就是其中之一。Cat C属半胱氨酸蛋白酶家族,具有独特的二肽肽酶活性,可以进行性地水解各种蛋白N末端的两个肽而参与多种蛋白修饰过程。在外周可激活杀伤性T淋巴细胞、肥大细胞和中性粒细胞中的丝氨酸蛋白酶(serineproteinase,SP),进而参与类风湿关节炎、哮喘、肺纤维化等多种炎症反应。特别是在有关动脉硬化研究中发现,Cat C不仅可以作为M1型巨噬细胞的标志物,还可通过自分泌作用影响巨噬细胞的极化,即CatC通过自分泌正反馈调节作用使巨噬细胞向M1方向极化。这个在外周系统炎症中非常重要的分子在神经系统的表达,是通过我们前期工作利用基因芯片查找CNS脱髓鞘疾病中差异表达的基因时发现的。并且,通过我们进一步研究发现CatC不仅在髓鞘脱失动物MG中高表达,也在脂多糖(Lipopolysaccharide,LPS)(5mg/kg)腹腔注射诱导的小鼠神经炎症模型的MG中高表达,说明CatC参与了神经炎症过程。然而CatC在神经炎症中的功能角色和参与神经炎症的机制不清。为阐述CatC在神经炎症中的功能角色,我们利用条件性CatC过表达(CatC over expression,Cat COE)和 CatC 敲减(CatC knock down,CatC KD)模式动物,将野生型(wild type,WT)同窝仔作为对照,利用LPS腹腔和脑室注射诱导神经炎症模型,通过比较不同基因型模型小鼠学习能力和脑内促炎性细胞因子的表达情况,阐明CatC的功能。再进一步通过体外实验,对CatC影响神经炎症的机制进行研究,并在体内实验中予以验证,以期对CatC影响神经炎症的机制进行探讨。实验材料和方法:1.实验对象及处理WT、条件性CatCOE和CatCKD三种基因型C57BL/6J背景小鼠,雌雄不限,体重在20～25g。在体内实验中,实验组以LPS注射(腹腔注射100μg/kg、侧脑室注射1 mg/kg)三种基因型小鼠,对照组(control)为0.9%生理盐水注射WT小鼠。在体外实验中,原代培养MG分别来自三种不同基因型小鼠,对照组(control)加2%FBS培养基,实验组以LPS(50ng/ml)或CatC活性单体(1、10、100 ng/ml)浓度梯度刺激。常规灌流取脑、冰冻切片,使用试剂盒提取组织和细胞的蛋白质、Trizol提取RNA。2.水迷宫定位航行实验Morris水迷宫用于小鼠学习能力行为学分析。不同基因型动物LPS腹腔注射后24小时开始定位航行实验,记录连续5天的游泳速度及逃避潜伏期。3.免疫组织化学染色和免疫细胞化学染色分别使用未处理、水迷宫实验结束及LPS侧脑室注射24小时小鼠脑组织冰冻切片,利用Ibal抗体检测MG形态;分别利用IL-1β及Cat C抗体检测促炎性细胞因子及Cat C表达;使用Cat C刺激24小时的原代培养WT MG,利用CD86抗体检测M1表达。4.原位杂交分别使用上述脑组织的冰冻切片,利用TNF-α、IL-1β探针检测脑内促炎性细胞因子的表达情况;利用c-fins及Cat C探针检测脑内MG活化及Cat C表达情况。5.酶联免疫吸附实验采用双抗体夹心法检测未处理、水迷宫实验结束时及LPS侧脑室注射24小时小鼠脑内及水迷宫实验结束时血清中的TNF-α、IL-lβ水平,以探讨促炎性细胞因子表达情况。6.Realtime-PCRRealtime-PCR检测上述小鼠脑内和Cat C刺激原代培养MG中促炎性细胞因子TNF-α、IL-lβ表达和MG极化情况。采用CD86、CD16、CD32、IL-6引物检测M1特异性标记分子的表达。7.流式细胞术流式细胞术用于Cat C活性单体1 OOng/ml刺激原代培养WT MG 24小时后MG极化检测。其中M1采用CD86-FITC抗体标记、M2采用CD206-APC抗体标记,使用CD16/32抗体封闭Fc段、同型对照消除非特异性结合。8.数据分析与处理实验结果以均数±标准差表示,应用SPSS19.0统计学软件对水迷宫数据采用重复测量数据的多因素方差分析和多变量方差分析;其他实验数据采用单因素方差分析和线性回归分析。P<0.05作为有统计学意义的判定标准。结果:1.Cat C基因差异表达对LPS诱导神经炎症小鼠学习能力的影响(1)不同基因类型小鼠脑内MG形态及促炎性细胞因子表达无显著差异。未做处理时,MG形态学显示WT和CatCKD组小鼠MG处于静息状态,CatC OE略活化;ELISA检测三种基因型小鼠脑组织匀浆TNF-α、IL-lβ的表达无显著性差异,提示在未做任何处理的情况下,CatC基因模式动物中不存在自发性神经炎症及其他炎性相关改变。(2)CatC参与低剂量LPS腹腔注射诱导的神经炎症LPS腹腔注射WT小鼠全脑内MG活化,TNF-α、IL-1β高表达,表明小剂量LPS(100μg/kg)腹腔注射引起了脑内神经炎症。同时,生理情况下只表达于海马CAⅡ区的Cat C阳性信号遍布于全脑,提示Cat C参与神经炎症。(3)CatCOE加重神经炎症小鼠学习能力损害水迷宫定位航行实验表明,三种基因型神经炎症小鼠与对照组之间平均游泳速度无显著差异,提示低剂量LPS注射未影响小鼠的运动能力;神经炎症小鼠从第2天开始潜伏期明显增加,提示低剂量LPS注射可造成其学习能力下降。其中,Cat C OE小鼠在第五天的潜伏期明显长于Cat C KD组,说明Cat C OE促进低剂量LPS介导的小鼠学习能力损害。(4)CatCOE加重炎症反应定位航行实验结束后检测到CatCOE小鼠MG剧烈活化、脑内TNF-α、IL-1β和血清内TNF-α表达升高最显著,WT组次之、Cat CKD组最低。提示Cat COE加重体内及脑内炎症反应,可能是导致学习功能受损的主要原因。Cat CKD则逆转了此过程。2.MG内Cat C差异表达对MG活化和促炎性细胞因子产生的影响(1)LPS侧脑室注射诱导脑内神经炎症为了排除外周系统炎症对脑内神经炎症的影响,我们采取LPS侧脑室注射的方法来建立神经炎症模型。模型小鼠全脑内MG活化、TNF-α、IL-1β表达显著增加,表明神经炎症模型诱导成功,可用于以下的实验研究。(2)神经炎症诱导CatC在脑内表达增加神经炎症小鼠MG表达的Cat C显著增加,表明Cat C参与了LPS侧脑室注射诱导的神经炎症。(3)CatCOE加重LPS侧脑室注射诱导的神经炎症LPS处理后,与Cat C KD相比,Cat C OE小鼠脑内MG活化最剧烈,TNF-α、IL-1β表达上调最显著,WT次之。提示脑内CatC上调加重LPS介导的神经炎症反应,下调则减轻。3.Cat C促进MG向M1极化加重神经炎症(1)Cat C影响LPS诱导的原代MG活化程度和促炎性细胞因子的产生给予LPS刺激三种基因型原代培养MG,Cat C OE组MG活化形态学变化最剧烈、促炎性细胞因子表达最高,WT组次之,CatCKD组最低。提示CatC上调促进了LPS介导的原代培养MG的活化,同时促进促炎性细胞因子的表达;下调则相反。(2)CatC促进原代培养MG向M1极化Cat C活性单体刺激WT原代MG M1标志物表达显著增高,并呈剂量依赖趋势,且M1染色阳性;流式细胞术检测CatC刺激后M1型MG数量显著增多,提示CatC促进了原代培养MG向M1极化。(3)CatC促进体内MG向M1极化在LPS脑室注射诱导神经炎症的体内实验中,与WT神经炎症小鼠相比,CatC OE小鼠脑内多种M1型MG的特异性标志物和促炎性细胞因子表达明显上调,而在CatCKD小鼠则抵消了这一影响,提示CatC在体内促进神经炎症MG向M1极化。结论:1.CatC过表达,加重LPS介导的神经炎症小鼠学习障碍。2.MG内Cat C过表达/敲减,加剧/减轻LPS介导的神经炎症反应。3.CatC通过促进MG向M1型极化加重神经炎症。更多还原

【Abstract】 Objective:Neuroinflammation is a common pathological feature of central nervous system(CNS)injury and diseases.The main pathological changes of neuroinflammation include overactivation of glial cells,infiltration of inflammatory cells,overproduction of proinflammatory cytokines,increased blood-brain-barrier permeability and so on.Microglia(MG)which is the immune cell and serves as the first line of defense of innate immunity in the CNS is first activated and plays an important role in the initial stage and progression of neuroinflammation.Activated MG can be polarized into M1 and M2 phenotypes with very different morphology and function.M1 phenotype which is also named classical activation can eliminate pathogen while producing proinflammatory cytokines such as TNF-a(tumor necrosis factor-a,TNF-a,IL-lp(interleukin-1β,IL-1β)eta that can aggravate inflammation and tissue damage.M2 phenotype which is also named alternative activation,can clear cell debris and release a large number of protective factors,such as IL-10,IL-4 and IGF-I(insulin-like growth factor 1,IGF-I).These factors can promote the reconstruction and repairment of damaged tissue.Numerous studies have shown closely relationship between the polarization of MG and various CNS diseases.There are significant differences in the phenotypic characteristics of MG in different disease or distinct stages in the same disease.Finding the reason of polarization and controlling polarization state may become a new therapeutic strategy of CNS disease.The activation of MG in neuroinflammation generally accompanies expression changes of many genes.Among these genes cathepsin C(Cat C)is one of them which was found in our previous study.CatC belongs to cystein protease family and has a unique aminodipeptidyl peptidase activity which can progressively remove dipeptides from N-terminal of protein,therefore participates in variety of protein modification proceses.In the peripheral system,it is reported that Cat C is involved in rheumatoid arthritis,asthma,pulmonary fibrosis and many other inflammatory reactions by activating the serine proteases(SP)in cytotoxic T lymphocytes,mast cells and neutrophils.Especially in the relevant study for arteriosclerosis,it is found that Cat C is not only the marker of M1 but also affects macrophage polarization through macrophages autocrine,namely Cat C regulates macrophages polarization into M1 through macrophages autocrine positive feedback manner.The CNS expression of Cat C which has been considered as a very important molecule in the peripheral system inflammation was found in our preliminary work aiming to find out the differentially expressed genes in demyelinating diseases by gene chip.The further study showed that Cat C was highly expressed in activated MG in both demyelinating model and LPS(lipopolysaccharide,LPS)(5 mg/kg)induced neuroinflammation,indicating that Cat might participat in the process of neuroinflammation.However,the functional role and mechanism of Cat C involved in the neuroinflammation is unclear.In order to clarify the functional role of Cat C in neuroinflammation,we used conditional Cat C OE(Cat C over expression,Cat C OE)and Cat C KD(Cat C knock down,Cat C KD)transgenic mice and littermate wild type(WT)mice as the control.Intraperitoneal and intracerebroventricular injection of LPS were used to induce neuroinflammation in the brain,then the learning ability behavior and expression of proinflammatory cytokines were analyzed in different genotype mice and compared to each other.The mechanism of Cat C involved in neuroinflammation was further studied by in vitro experiments,and was confirmed in vivo by using neuroinflammation animal model.Materials and methods:1.Animal and processing methodsFor the in vivo experiments,C57BL/6J mice with genotypes of WT,conditional Cat C KD and Cat C OE weighing 20 to 25 g were used.In the experimental groups,mice with different genotype suffered LPS injection respectively(intraperitoneal injection 10Oug/kg or intracerebroventricular injection 1 mg/kg)to induce neuroinflammation of brain,and the WT mice were treated by equivolume 0.9%saline vehicle as the control group.For the in vitrol experiments,primary cultured MG were isolated from mice with different genotype then were incubated with LPS(50 ng/kg)or recombinant active Cat C(1,10,100 ng/ml)for 24 hours,and control MG isolated from WT mice were incubated with 2%FBS.Brain perfusion and frozen section were made as the common practice.The protein and RNA were extracted from tissue or culture cells by using reagent kit and trizol regent respectively.2.Navigation experiment of water mazeThe learning ability of mice was determined by Morris water maze test.After LPS intraperitoneal injection for 24 hours,the swimming speed and escape latency of different genotype mice were continuously recorded for 5 days.3.Immunohistochemical(IHC)and Immunocytochemical(ICC)stainingMG morphology was detected by Ibal IHC on the brain slides which come from the mice which are untreated or end of Morris water maze test or LPS intracerebroventricular injection for 24 hous.IL-Iβ and Cat C IHC were used to detect the expression of proinflammatory cytokines and ’Cat C respectively.After incubating with LPS for 24 hours,CD86 ICC which is Ml marker was performed in primary cultured MG from WT mice.4.In situ hybridization(ISH)The expression of proinflammatory cytokines mRNA were detected by TNF-a and IL-1(3 probes,the activation of MG and the expression of Cat C mRNA were detected by c-fins and Cat C probes by using the frozen brain sections mentioned before..5.Enzyme linked immunosorbent assay(ELISA)ELISA-sandwich technique was used to investigate TNF-a and IL-1β expression in brain and serum.For the expression in brain,the proteins were extracted from untreated,end of water maze test and LPS intracerebroventricular injection 24 hours mice respectively.For the expression of serum,the serum was collected at the end of water maze test.6.Realtime-PCRRealtime-PCR was used to detect the expression of TNF-aand IL-1β in above-mentioned mouse brain and the Ml polarization after primary cultured WT MG treated by recombinant active Cat C by using TNF-a,IL-lβ,CD86,CD 16,CD32 and IL-6 primers.7.Flow cytometryFlow cytometry was used to detect MG polarization after stimulating with recombinant active Cat C 100ng/ml for 24 hours.CD86-FITC and CD206-APC were used for M1 and M2 detection,and CD16/32 was used for blocking Fc receptor to minimize nonspecific binding.8.Statistical analysisThe experimental results were expressed as mean ± standard error of the mean(SEM).Repeated measures multivariate ANOVA and multivariate analysis of variance were used for water maze test results analysis.Other data were evaluated for one-way ANOVA and linear regression.All statistical analyses were performed in the Statistical Package for Social Sciences 19.0.Differences with P<0.05 were considered as statistically significant difference.Results:1.The impact of Cat C differentiated expression on learning ability in LPS-induced neuroinflammation(1)The morphology of MG and the expression of proinflammatory cytokines had no significantly different in different genotype miceIn untreated position,the MG in Cat C KD and WT mice were in resting state,in Cat C OE mice were slightly activated.There were no significant differences between the three genotype mice in the expression of TNF-α and IL-1β in the brain.These results indicated that no spontaneous neuroinflammation and other inflammatory changes occurring in untreated conditional Cat C transgenic mice.(2)Cat C was involved in low dose of LPS intraperitoneal injection induced neuroinflammationAfter LPS injection,MG were activated in the whole brain,and TNF-α and IL-1βwere significantly upregulated in WT mice after LPS(100μg/kg)intraperitoneal injection.This result suggested that low dose of LPS intraperitoneal injection could induce neuroinflammation in mouse brains.Meanwhile,Cat C positive signals were found in the whole brain which were only found in the CAII region of hippocampus under physiological conditions,suggesting that Cat C is involved in the neuroinflammatory process.(3)Cat OE aggravated impairment of learning ability in neuroinflammatory miceMorris Water Maze test showed there was no difference between the three genotype mice on swimming speed in the navigation experiment,suggesting that the motor ability of the mice was not affected following LPS treatment.However,the escape latency of neuroinflammatory mice increased significantly from the second days,which indicated that the low dose of LPS injection could cause the decline of learning ability,and the scape latency was significantly longer in Cat C OE mice than that in Cat C KD mice on the fifth day,indicating that Cat C OE aggravated the impairment of learning ability following low dose LPS treatment.(4)Cat C OE aggravated inflammatory reactionAt the end of navigation experiment of water maze,the mice were sacrificed to detect the expression of TNF-a,IL-1(3 in brain and IL-1β in serum by ELISA.The results showed that the highest expression levels of these inflammatory factors were found in Cat C OE mice,then followed by WT mice,and the lowest were found in Cat CKD mice.These results indicate that Cat C OE aggravated the inflammatory response both in peripheral blood and brain,which might be the main causes of impaired learning ability.Cat C KD reversed this process.2.The impact of microglial Cat C differentiated expression on the activation of MG and expression of proinflammatory cytokines(1)LPS intracerebroventricular injection induced neuroinflammationIn order to eliminate the influence of systemic inflammation on the brain,we adopted LPS intracerebroventricular injection to establish neuroinflammation model.The activation of MG and the upregulated expression of TNF-a and IL-1β were found in the whole brain of mice,indicating the neuroinflammation model was successful established and can be used for further study.(2)Neuroinflammation induced the high expression of Cat CThe expression of microglial Cat C was significantly increased in neuroinflammation mouse brain,indicating that Cat C was involved in the induction of neuroinflammation following LPS intracerebroventricular injection.(3)Cat C OE aggravated neuroinflammation induced by LPS intracerebroventri-cular injection.After the mice were treated by LPS intracerebroventricular injection,severely activated MG pattern was found in Cat C OE mouse brain.Meanwhile,in Cat C OE mouse brain the expression of proinflammatory cytokines TNF-a and IL-1β increased significantly at the highest level compared with that in WT mice,and the lowest level was found in Cat C KD mice.These results indicated that Cat C OE aggravated LPS mediated neuroinflammatory reaction.In contrast,Cat C KD reduced this reaction.3.Cat C promoted MG polarization into M1 phenotype and aggravated neuroinflammation(1)Cat C affected on the activation status and the production of proinflammatory cytokines in LPS-treated primary cultured MG.The morphology of the three genotype primary cultured MG was similar.After LPS treatment,severely activated MG pattern was found in Cat C OE group,meanwhile the expression of proinflammatory cytokines TNF-a and IL-lβ also increased significantly at the highest level.In constrast,Cat C KD MG showed that at the lowest level.It suggest that Cat C OE enhanced the activation of primary cultured MG after LPS stimulation and production of proinflammatory cytokines,however,Cat C KD reduced them.(2)Cat C promoted primary cultured MG to polarize into Ml phenotypeIt was found the expression of M1 mRNA markers in primary cultured WT MG stimulated by recombinant active Cat C was elevated in a dose dependent manner,and M1 marker positive cells was also detected.The number of Ml phenotype MG was significantly increased following recombinant active Cat C stimulation by flow cytometry analysis.The results suggests that Cat C has the function that promoted the primary cultured MG to polarize into Ml phenotype.(3)Cat C promoted MG to polarize into Ml phenotype in vivoThe in vitro experiments were performed again in in vivo experiments.The results showed the specific markers of Ml and proinflammatory cytokines mRNA were significantly up-regulated in Cat C OE mice compared with that in WT mice treated by LPS,whereas down-regulated in Cat C KD,indicating that Cat C promoted MG to polarize into Ml phenotype in vivo.Conclusions:1.Cat C overexpression aggravates impairment of spatial learning ability in LPS-induced neuroinflammatory mice.2.Microglial Cat C OE/KD promotes/inhibits LPS-induced neuroinflammation.3.Cat C aggravates neuroinflammation by promoting microglial M1 polarization.更多还原