本文選題：鸚鵡熱嗜衣原體　切入點(diǎn)：LIGHT　出處：《南華大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討LIGHT信號通路在鸚鵡熱嗜衣原體(Chlamydia psittaci,Cps)呼吸道感染過程中的作用及機(jī)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,每只小鼠鼻內(nèi)接種4.5×105 IFU Cps,每天觀察記錄小鼠的進(jìn)食、活動、體重變化及生存情況。在感染前和感染后第4、9、14天分別處死各組8只小鼠,其中4只小鼠的肺組織用于制備肺組織勻漿,體外感染Hela細(xì)胞,通過間接免疫熒光法(indirect immunofluorescence assay,IFA)測定包涵體形成單位(inclusion forming unit,IFU);另4只小鼠的右肺用于制備支氣管肺泡灌洗液(broncho-alveolar lavage fluid,BALF)并對其中浸潤的細(xì)胞進(jìn)行計數(shù),部分左肺經(jīng)多聚甲醛固定,用于制作石蠟切片并進(jìn)行蘇木素-伊紅染色(hematoxylin and eosin Staining,HE),以評估肺部病理損害,剩余部分左肺用于提取總RNA,通過逆轉(zhuǎn)錄-定量PCR法(reverse transcription-quantitative polymerase chain reaction,RT-q PCR)檢測IFN-γ、TNF-α、IL-17、IL-4、IL-6和IL-12的m RNA水平。分離小鼠脾臟,制備脾細(xì)胞懸液,流式細(xì)胞術(shù)(Flow Cytometry,FCM)檢測脾臟中CD4+T細(xì)胞、CD8+T細(xì)胞和調(diào)節(jié)性T細(xì)胞(Regulatory T cell,Treg)的百分率。結(jié)果:經(jīng)呼吸道感染Cps后,小鼠出現(xiàn)進(jìn)食減少、活動下降、體重減輕等癥狀,并出現(xiàn)死亡。與WT小鼠相比,LIGHT KO小鼠上述癥狀出現(xiàn)更嚴(yán)重,并有更高的死亡率。肺組織病理結(jié)果顯示,Cps感染導(dǎo)致肺部出現(xiàn)充血、水腫和炎性細(xì)胞浸潤等病理改變,LIGHT KO小鼠的肺病理損害程度顯著重于WT小鼠。與之對應(yīng),LIGHT KO小鼠BALF中浸潤的炎性細(xì)胞數(shù)量顯著多于WT小鼠。IFA結(jié)果顯示,感染Cps后的第4、9、14天,LIGHT KO小鼠的肺部Cps載量均顯著高于WT小鼠;至感染后第14天,WT小鼠肺部Cps已被清除,而LIGHT KO小鼠肺部仍可檢出Cps。兩組小鼠脾細(xì)胞中CD4+T細(xì)胞和CD8+T細(xì)胞百分率和比例無明顯區(qū)別,但LIGHT KO小鼠的Treg細(xì)胞比例顯著高于WT小鼠。兩組小鼠感染Cps后細(xì)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小鼠這些細(xì)胞因子水平均顯著低于WT小鼠。結(jié)論:在Cps呼吸道感染過程中,LIGHT信號通路缺失可通過促進(jìn)Treg細(xì)胞分化,并抑制與T細(xì)胞應(yīng)答相關(guān)的IFN-γ、TNF-α、IL-17等細(xì)胞因子產(chǎn)生,而影響機(jī)體抗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- 緯 -TNF- 偽 -TNF- 偽 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- 緯 TNF- 偽 and IL-17 related to T cell response. The immune effect of Cps infection was affected.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R563

【共引文獻(xiàn)】

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本文編號：1617607