CD157在结核患者体内的表达水平、作用及其机制的初步探讨

发布时间：2018-05-14 00:12

本文选题：CD157 + 结核分枝杆菌　；参考：《蚌埠医学院》2015年硕士论文

【摘要】：目的:1.检测健康人群、结核潜伏感染者、肺炎患者、肺结核患者外周血单个核细胞和血浆中CD157水平,了解CD157在这些人群体内的表达情况。2.利用体外细胞模型(结核分枝杆菌H37Ra感染人外周血的单核巨噬细胞系THP-1、外周血单个核细胞)探讨CD157表达增高的作用及其相关机制。方法:1.利用q PCR检测各组人群外周血单个核细胞(PBMC)中CD157 m RNA的表达水平;利用ELISA检测血浆和配对胸水上清中游离CD157的蛋白水平;流式细胞术分析CD157在不同细胞表面的表达情况。2.收集结核性胸膜炎和非结核性胸膜炎患者胸腔积液,用ELISA检测上清中游离CD157蛋白水平。3.对部分活动性肺结核患者进行随访,检测其治疗前及治疗后3、6、12月外周血PBMC中CD157的m RNA水平,以及血浆中游离CD157浓度的变化情况。4.利用结核分枝杆菌(结核杆菌)H37Ra感染THP-1和PBMC细胞,检测细胞中CD157的表达情况。5.检测结核性胸膜炎患者胸水中CD157和γ干扰素(IFN-γ)表达情况,比较两者相关性;用结核性胸膜炎患者和非结核性胸膜炎患者胸水培养THP-1细胞,加入IFN-γ或中和IFN-γ抗体,检测其对CD157表达的影响。6.用CD157和结核杆菌作用于THP-1细胞,观察CD157对结核杆菌感染的巨噬细胞分泌趋化因子的能力及其对细胞迁移的影响。结果:1.CD157在肺结核患者体内表达升高:(1)在PBMC中,肺结核患者外周血单个核细胞中CD157m RNA的表达水平显著高于健康对照组、结核潜伏感染组和肺炎患者组(P0.05);流式结果显示,CD157主要表达于单核细胞以及中性粒细胞表面,T细胞表面几乎不表达,但在肺结核患者体内表达CD157的单核细胞和中性粒细胞的比例与健康对照组相比并没有显著性差异;在血浆中,肺结核患者血浆游离CD157浓度显著高于结核潜伏感染组、肺炎对照组和健康对照组(P0.05);而CD157 m RNA在痰涂片阳性和痰涂片阴性的活动性肺结核患者中的表达水平并没有显著性差异。2.CD157的表达水平随着抗结核治疗降低:检测治疗后3、6、12月活动性肺结核患者的外周血中CD157的m RNA水平,以及血浆中游离CD157浓度,结果显示,肺结核患者治疗后6个月和12个月外周血中CD157 m RNA表达水平和血浆中CD157浓度显著降低,相比治疗前差异具有统计学意义(P0.05)。3.CD157在结核性胸膜炎患者胸水中表达增高:CD157 m RNA和游离CD157在结核性胸膜炎患者胸水中的表达水平显著高于其在外周血中的表达水平。结核性胸膜炎患者胸水的游离CD157的含量显著高于非结核性胸膜炎患者(P0.05)。4.CD157可诱导结核杆菌感染的巨噬细胞分泌CCL2,进而促进细胞迁移:利用CD157和结核杆菌共作用于THP-1细胞,结果显示THP-1细胞中CCL2的表达显著高于单独的CD157或者结核杆菌作用的细胞(P0.05);进一步的细胞迁移(Transwell)实验表明,CD157和结核杆菌共作用的THP-1细胞上清可以显著提高细胞的迁移率,而在细胞上清中加入抗CCL2的中和抗体,这种迁移作用得到了显著的抑制,表明CD157是通过促进结核杆菌感染的巨噬细胞中CCL2的表达来调节细胞的迁移。5.结核杆菌通过上调IFN-γ诱导CD157的表达:结核杆菌H37Ra感染THP-1和PBMC细胞的CD157 m RNA或细胞培养上清中游离CD157表达量并不增高,与对照相比,差异没有统计学意义(P0.05)。利用结核性胸膜炎患者和非结核性胸膜炎患者胸水作用于THP-1细胞,结果发现结核性胸膜炎患者胸水作用的THP-1细胞中,CD157 m RNA的相对表达水平以及游离CD157的浓度都显著高于非结核性胸膜炎患者胸水作用的细胞(P0.05)。进一步,我们发现肺结核患者中IFN-γ和CD157的表达成正相关。因此我们利用功能性IFN-γ或者IFN-γ中和抗体作用THP-1细胞,结果发现IFN-γ可以显著提高细胞中CD157的表达(P0.05)。结论:1.CD157在肺结核患者外周血中表达增高,其表达水平随着抗结核治疗逐步下降。2.CD157可以促进结核杆菌感染的巨噬细胞分泌CCL2,进而促进细胞迁移。3.结核性胸膜炎患者胸水中,IFN-γ与CD157的表达水平呈正相关。IFN-γ可以诱导巨噬细胞CD157的表达。
[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.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R52

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