發(fā)布時(shí)間：2018-05-14 00:12

  本文選題：CD157 + 結(jié)核分枝桿菌　； 參考：《蚌埠醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的:1.檢測健康人群、結(jié)核潛伏感染者、肺炎患者、肺結(jié)核患者外周血單個(gè)核細(xì)胞和血漿中CD157水平,了解CD157在這些人群體內(nèi)的表達(dá)情況。2.利用體外細(xì)胞模型(結(jié)核分枝桿菌H37Ra感染人外周血的單核巨噬細(xì)胞系THP-1、外周血單個(gè)核細(xì)胞)探討CD157表達(dá)增高的作用及其相關(guān)機(jī)制。方法:1.利用q PCR檢測各組人群外周血單個(gè)核細(xì)胞(PBMC)中CD157 m RNA的表達(dá)水平;利用ELISA檢測血漿和配對(duì)胸水上清中游離CD157的蛋白水平;流式細(xì)胞術(shù)分析CD157在不同細(xì)胞表面的表達(dá)情況。2.收集結(jié)核性胸膜炎和非結(jié)核性胸膜炎患者胸腔積液,用ELISA檢測上清中游離CD157蛋白水平。3.對(duì)部分活動(dòng)性肺結(jié)核患者進(jìn)行隨訪,檢測其治療前及治療后3、6、12月外周血PBMC中CD157的m RNA水平,以及血漿中游離CD157濃度的變化情況。4.利用結(jié)核分枝桿菌(結(jié)核桿菌)H37Ra感染THP-1和PBMC細(xì)胞,檢測細(xì)胞中CD157的表達(dá)情況。5.檢測結(jié)核性胸膜炎患者胸水中CD157和γ干擾素(IFN-γ)表達(dá)情況,比較兩者相關(guān)性;用結(jié)核性胸膜炎患者和非結(jié)核性胸膜炎患者胸水培養(yǎng)THP-1細(xì)胞,加入IFN-γ或中和IFN-γ抗體,檢測其對(duì)CD157表達(dá)的影響。6.用CD157和結(jié)核桿菌作用于THP-1細(xì)胞,觀察CD157對(duì)結(jié)核桿菌感染的巨噬細(xì)胞分泌趨化因子的能力及其對(duì)細(xì)胞遷移的影響。結(jié)果:1.CD157在肺結(jié)核患者體內(nèi)表達(dá)升高:(1)在PBMC中,肺結(jié)核患者外周血單個(gè)核細(xì)胞中CD157m RNA的表達(dá)水平顯著高于健康對(duì)照組、結(jié)核潛伏感染組和肺炎患者組(P0.05);流式結(jié)果顯示,CD157主要表達(dá)于單核細(xì)胞以及中性粒細(xì)胞表面,T細(xì)胞表面幾乎不表達(dá),但在肺結(jié)核患者體內(nèi)表達(dá)CD157的單核細(xì)胞和中性粒細(xì)胞的比例與健康對(duì)照組相比并沒有顯著性差異;在血漿中,肺結(jié)核患者血漿游離CD157濃度顯著高于結(jié)核潛伏感染組、肺炎對(duì)照組和健康對(duì)照組(P0.05);而CD157 m RNA在痰涂片陽性和痰涂片陰性的活動(dòng)性肺結(jié)核患者中的表達(dá)水平并沒有顯著性差異。2.CD157的表達(dá)水平隨著抗結(jié)核治療降低:檢測治療后3、6、12月活動(dòng)性肺結(jié)核患者的外周血中CD157的m RNA水平,以及血漿中游離CD157濃度,結(jié)果顯示,肺結(jié)核患者治療后6個(gè)月和12個(gè)月外周血中CD157 m RNA表達(dá)水平和血漿中CD157濃度顯著降低,相比治療前差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.CD157在結(jié)核性胸膜炎患者胸水中表達(dá)增高:CD157 m RNA和游離CD157在結(jié)核性胸膜炎患者胸水中的表達(dá)水平顯著高于其在外周血中的表達(dá)水平。結(jié)核性胸膜炎患者胸水的游離CD157的含量顯著高于非結(jié)核性胸膜炎患者(P0.05)。4.CD157可誘導(dǎo)結(jié)核桿菌感染的巨噬細(xì)胞分泌CCL2,進(jìn)而促進(jìn)細(xì)胞遷移:利用CD157和結(jié)核桿菌共作用于THP-1細(xì)胞,結(jié)果顯示THP-1細(xì)胞中CCL2的表達(dá)顯著高于單獨(dú)的CD157或者結(jié)核桿菌作用的細(xì)胞(P0.05);進(jìn)一步的細(xì)胞遷移(Transwell)實(shí)驗(yàn)表明,CD157和結(jié)核桿菌共作用的THP-1細(xì)胞上清可以顯著提高細(xì)胞的遷移率,而在細(xì)胞上清中加入抗CCL2的中和抗體,這種遷移作用得到了顯著的抑制,表明CD157是通過促進(jìn)結(jié)核桿菌感染的巨噬細(xì)胞中CCL2的表達(dá)來調(diào)節(jié)細(xì)胞的遷移。5.結(jié)核桿菌通過上調(diào)IFN-γ誘導(dǎo)CD157的表達(dá):結(jié)核桿菌H37Ra感染THP-1和PBMC細(xì)胞的CD157 m RNA或細(xì)胞培養(yǎng)上清中游離CD157表達(dá)量并不增高,與對(duì)照相比,差異沒有統(tǒng)計(jì)學(xué)意義(P0.05)。利用結(jié)核性胸膜炎患者和非結(jié)核性胸膜炎患者胸水作用于THP-1細(xì)胞,結(jié)果發(fā)現(xiàn)結(jié)核性胸膜炎患者胸水作用的THP-1細(xì)胞中,CD157 m RNA的相對(duì)表達(dá)水平以及游離CD157的濃度都顯著高于非結(jié)核性胸膜炎患者胸水作用的細(xì)胞(P0.05)。進(jìn)一步,我們發(fā)現(xiàn)肺結(jié)核患者中IFN-γ和CD157的表達(dá)成正相關(guān)。因此我們利用功能性IFN-γ或者IFN-γ中和抗體作用THP-1細(xì)胞,結(jié)果發(fā)現(xiàn)IFN-γ可以顯著提高細(xì)胞中CD157的表達(dá)(P0.05)。結(jié)論:1.CD157在肺結(jié)核患者外周血中表達(dá)增高,其表達(dá)水平隨著抗結(jié)核治療逐步下降。2.CD157可以促進(jìn)結(jié)核桿菌感染的巨噬細(xì)胞分泌CCL2,進(jìn)而促進(jìn)細(xì)胞遷移。3.結(jié)核性胸膜炎患者胸水中,IFN-γ與CD157的表達(dá)水平呈正相關(guān)。IFN-γ可以誘導(dǎo)巨噬細(xì)胞CD157的表達(dá)。
[Abstract]:Objective: 1. to detect the level of CD157 in peripheral blood mononuclear cells and plasma in healthy people, patients with latent tuberculosis, pneumonia, and pulmonary tuberculosis, and to understand the expression of CD157 in these population by.2. using in vitro cell model (mononuclear macrophage THP-1, peripheral blood mononuclear cells) in human peripheral blood infected by Mycobacterium tuberculosis (MTB H37Ra) To investigate the role of CD157 expression and its related mechanisms. Methods: 1. the expression level of CD157 m RNA in peripheral blood mononuclear cells (PBMC) was detected by Q PCR, and the protein level of free CD157 in plasma and paired thoracic water supernatant was detected by ELISA, and the expression of CD157 on the surface of different cells was analyzed by flow cytometry. The pleural effusion of patients with nuclear pleurisy and non tuberculous pleurisy was followed up by ELISA detection of free CD157 protein level.3. in the supernatant to detect the m RNA level of CD157 in PBMC of peripheral blood of 3,6,12 months before and after treatment, and the change of free CD157 concentration in plasma.4. using tuberculosis branch rod. Bacteria (Mycobacterium tuberculosis) H37Ra infected THP-1 and PBMC cells, and the expression of CD157 in the cells was detected by.5. to detect the expression of CD157 and interferon gamma (IFN- gamma) in the pleural effusion of tuberculous pleurisy, and the correlation was compared; THP-1 cells in the pleural effusion of patients with tuberculous pleurisy and non tuberculous pleurisy were cultured with IFN- gamma or neutralization IFN- gamma. The effect of.6. on the expression of CD157 and the effect of CD157 and Mycobacterium tuberculosis on THP-1 cells to observe the ability of CD157 to secrete chemokines from Mycobacterium tuberculosis and its effect on cell migration. Results: 1.CD157 is elevated in the body of tuberculosis patients: (1) in PBMC, CD15 in peripheral blood mononuclear cells of tuberculosis patients The expression level of 7m RNA was significantly higher than that in the healthy control group, the latent tuberculosis group and the pneumonia patient group (P0.05), and the flow results showed that CD157 was mainly expressed in monocyte and neutrophil surface, and the surface of T cells was almost not expressed, but the proportion of CD157 mononuclear cells and neutrophils in the patients with pulmonary tuberculosis was compared with that of health. The plasma free CD157 concentration in the plasma was significantly higher than that of the latent tuberculosis group, the pneumonia control group and the healthy control group (P0.05), while the expression level of CD157 m RNA in sputum smear positive and sputum negative active pulmonary tuberculosis patients had no significant difference of.2.CD157 expression water. The decrease in anti tuberculosis treatment: the m RNA level of CD157 in the peripheral blood of 3,6,12 month active pulmonary tuberculosis patients after treatment and the concentration of free CD157 in plasma. The results showed that the CD157 m RNA expression level and the concentration of CD157 in plasma were significantly reduced in 6 and 12 months after treatment, compared with pre treatment differences. There was a statistically significant (P0.05).3.CD157 expression in the pleural effusion of patients with tuberculous pleurisy: the expression level of CD157 m RNA and free CD157 in the pleural effusion of patients with tuberculous pleurisy was significantly higher than that in peripheral blood. The free CD157 content of pleural effusion in patients with tuberculous pleurisy was significantly higher than that of non tuberculous pleurisy patients (P0). .05).4.CD157 could induce the secretion of CCL2 from macrophages infected by Mycobacterium tuberculosis, and then promote cell migration: the use of CD157 and Mycobacterium tuberculosis co acted on THP-1 cells. The results showed that the expression of CCL2 in THP-1 cells was significantly higher than that of single CD157 or Mycobacterium tuberculosis (P0.05); further cell migration (Transwell) experiments showed CD157. The THP-1 cell supernatant co acted with Mycobacterium tuberculosis could significantly increase the cell migration rate, and the anti CCL2 neutralization antibody was added to the cell supernatant. This migration effect was significantly inhibited, indicating that CD157 was used to regulate the migration of.5. tuberculosis through the expression of CCL2 in macrophages infected by Mycobacterium tuberculosis. IFN- gamma induced CD157 expression: the expression of free CD157 in CD157 m RNA or cell culture supernatant was not increased in THP-1 and PBMC cells infected by Mycobacterium tuberculosis. Compared with the control, the difference was not statistically significant (P0.05). The effect of tuberculosis on tuberculous pleuritis and non tuberculous pleuritis was found in THP-1 cells, and tuberculosis was found to be tuberculosis. In THP-1 cells with pleural effusion, the relative expression level of CD157 m RNA and the concentration of free CD157 are significantly higher than those of non tuberculous pleurisy patients (P0.05). Further, we found that the expression of IFN- gamma and CD157 in the patients with pulmonary tuberculosis is positively correlated. Therefore, we use functional IFN- gamma or IFN- gamma. Neutralizing antibodies acting on THP-1 cells, the results showed that IFN- gamma could significantly increase the expression of CD157 in the cells (P0.05). Conclusion: the expression of 1.CD157 in the peripheral blood of tuberculosis patients is increased. The expression level of 1.CD157 can promote the secretion of CCL2 in the macrophages infected by tuberculosis with the gradual decrease of.2.CD157 in the anti tuberculosis treatment, and thus promote the migration of.3. in the cells. In patients with pleurisy, the expression level of IFN- gamma was positively correlated with CD157 expression..IFN- CD157 could induce the expression of macrophage CD157.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R52

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本文編號(hào)：1885444