LIGHT在鹦鹉热嗜衣原体呼吸道感染中的作用及机制研究

发布时间：2018-03-16 01:02

本文选题：鹦鹉热嗜衣原体　切入点：LIGHT　出处：《南华大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:探讨LIGHT信号通路在鹦鹉热嗜衣原体(Chlamydia psittaci,Cps)呼吸道感染过程中的作用及机制。方法:野生型(Wild type,WT)小鼠和LIGHT基因敲除(Knock out,KO)小鼠各50只,每只小鼠鼻内接种4.5×105 IFU Cps,每天观察记录小鼠的进食、活动、体重变化及生存情况。在感染前和感染后第4、9、14天分别处死各组8只小鼠,其中4只小鼠的肺组织用于制备肺组织匀浆,体外感染Hela细胞,通过间接免疫荧光法(indirect immunofluorescence assay,IFA)测定包涵体形成单位(inclusion forming unit,IFU);另4只小鼠的右肺用于制备支气管肺泡灌洗液(broncho-alveolar lavage fluid,BALF)并对其中浸润的细胞进行计数,部分左肺经多聚甲醛固定,用于制作石蜡切片并进行苏木素-伊红染色(hematoxylin and eosin Staining,HE),以评估肺部病理损害,剩余部分左肺用于提取总RNA,通过逆转录-定量PCR法(reverse transcription-quantitative polymerase chain reaction,RT-q PCR)检测IFN-γ、TNF-α、IL-17、IL-4、IL-6和IL-12的m RNA水平。分离小鼠脾脏,制备脾细胞悬液,流式细胞术(Flow Cytometry,FCM)检测脾脏中CD4+T细胞、CD8+T细胞和调节性T细胞(Regulatory T cell,Treg)的百分率。结果:经呼吸道感染Cps后,小鼠出现进食减少、活动下降、体重减轻等症状,并出现死亡。与WT小鼠相比,LIGHT KO小鼠上述症状出现更严重,并有更高的死亡率。肺组织病理结果显示,Cps感染导致肺部出现充血、水肿和炎性细胞浸润等病理改变,LIGHT KO小鼠的肺病理损害程度显著重于WT小鼠。与之对应,LIGHT KO小鼠BALF中浸润的炎性细胞数量显著多于WT小鼠。IFA结果显示,感染Cps后的第4、9、14天,LIGHT KO小鼠的肺部Cps载量均显著高于WT小鼠;至感染后第14天,WT小鼠肺部Cps已被清除,而LIGHT KO小鼠肺部仍可检出Cps。两组小鼠脾细胞中CD4+T细胞和CD8+T细胞百分率和比例无明显区别,但LIGHT KO小鼠的Treg细胞比例显著高于WT小鼠。两组小鼠感染Cps后细胞因子IFN-γ、TNF-α、IL-17、IL-4、IL-6及IL-12的m RNA水平均有升高,且LIGHT KO小鼠这些细胞因子水平均显著低于WT小鼠。结论:在Cps呼吸道感染过程中,LIGHT信号通路缺失可通过促进Treg细胞分化,并抑制与T细胞应答相关的IFN-γ、TNF-α、IL-17等细胞因子产生,而影响机体抗Cps感染免疫作用。
[Abstract]:Objective: to investigate the role and mechanism of LIGHT signaling pathway in the process of respiratory tract infection of Chlamydia psittacidia psittacidia psittacidia plamydia psittaciae. Methods: wild type WTT mice and LIGHT gene knockout Knock outKO50 mice were used in this study. Each mouse was inoculated intranasally with 4.5 脳 10 ~ 5 IFU CPS. The feeding, activity, weight change and survival of the mice were observed and recorded every day. Eight mice in each group were killed before infection and on the 14th day after infection. The lung tissues of 4 mice were used to prepare lung tissue homogenate and infect Hela cells in vitro. Indirect immunofluorescence assay (IFA) was used to determine inclusion forming unit, the right lung of the other four mice was used to prepare broncho-alveolar lavage fluido (BALF) and count the infiltrating cells, and some of the left lungs were fixed by polyformaldehyde. To make paraffin sections and make hematoxylin and eosin staininghehe for hematoxylin and eosin staining to evaluate lung pathological damage. The rest of the left lung was used to extract total RNAs. Reverse transcription-quantitative polymerase chain reactionation was performed by reverse transcription-quantitative PCR method to detect the m RNA levels of IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, IL 4, and IL-12 in the spleen of mice. Flow Cytometry (FCM) was used to detect the percentage of CD4 T cell CD8 T cells and regulatory T cell T cell TregT cells in the spleen. Results: after respiratory tract infection with Cps, the mice developed symptoms such as decreased food intake, decreased activity and weight loss. Compared with WT mice, LIGHT KO mice had more severe symptoms and higher mortality. The pathological results of lung tissue showed that CPS infection caused congestion in the lungs. The pathological changes such as edema and inflammatory cell infiltration in the lung of LIGHT KO mice were more severe than those in WT mice, and the number of infiltrating inflammatory cells in BALF of LIGHT KO mice was significantly higher than that of WT mice. The lung Cps load of LIGHT KO mice was significantly higher than that of WT mice on the 14th day after infection with Cps, and the lung Cps of WT mice had been cleared by 14 days after infection. However, the percentage and proportion of CD4 T cells and CD8 T cells in spleen cells of LIGHT KO mice could still be detected in the lungs of the two groups, and there was no significant difference in the percentage and proportion of CD4 T cells and CD8 T cells between the two groups. However, the proportion of Treg cells in LIGHT KO mice was significantly higher than that in WT mice. The levels of cytokine IFN- 纬 -TNF- 伪 -TNF- 伪 IL-17 IL-4, IL-6 and m RNA of IL-12 were increased in both groups. The levels of these cytokines in LIGHT KO mice were significantly lower than those in WT mice. Conclusion: the absence of light signaling pathway in Cps respiratory tract infection can promote the differentiation of Treg cells and inhibit the production of cytokines such as IFN- 纬 TNF- 伪 and IL-17 related to T cell response. The immune effect of Cps infection was affected.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R563

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