肺结核T细胞耗竭的临床观察及动物模型治疗的实验研究

发布时间：2018-04-19 20:38

本文选题：结核病 + 空洞型肺结核　；参考：《兰州大学》2017年硕士论文

【摘要】：全世界范围内,结核病的感染率和致死率都非常高,尤其是在中国和印度等发展中国家,结核病给社会以及个人带来了沉重的负担。在结核分枝杆菌感染的初期,大多数人能通过自身免疫力阻止结核病的进一步发展,大约10%的人群最终发展成活动性肺结核。在结核分枝杆菌(Mtb)感染的后期,机体免疫系统由于受到损伤而导致结核分枝杆菌复苏,形成慢性感染性结核,机体在长期慢性感染的作用下,主要表现为免疫功能低下。重症肺结核病人为何表现为免疫力低下?是否发生了T细胞功能耗竭?本研究通过分析临床肺结核病人外周血T细胞功能耗竭相关的几组功能学指标,来探讨慢性肺结核病人发生T细胞耗竭的机制。目的为分析慢性肺结核病人免疫功能低下的原因,探讨结核分枝杆菌在人体内的持续感染是否造成T细胞功能耗竭,研究持续痰菌阳性的空洞性肺结核病人T细胞功能的变化。方法:抽取兰州市肺科医院正在住院治疗的肺结核病人51例及健康志愿者16例,分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)。1,利用酶联免疫斑点试验(ELISPOT)检测外周血T淋巴细胞经抗原ESAT-6和CFP-10刺激后IFN-γ的分泌水平;2,利用ELISA技术检测T淋巴细胞培养基上清中IL-2的分泌水平;3,在结核菌素纯蛋白衍生物(PPD)的刺激下培养PBMCs,用流式细胞术分析外周血T细胞的增殖能力;4,检测CD4~+、CD8~+T细胞的数目,以及CD4~+、CD8~+T细胞表面免疫抑制相关分子T细胞免疫球蛋白粘蛋白分子3(T cell immunoglobulin mucin domain containingmolecules 3,TIM-3)的表达;5,调取各组病人同一时期外周血血象,分析外周血中红细胞、血红蛋白、白细胞、血小板以及单个核细胞结果;6,通过分析病人性别、年龄、病程、初(复)治、痰抗酸染色、肺部影像学检查结果与T细胞耗竭相关指标的关系,来探究几种类型的慢性肺结核病人发生T细胞耗竭的情况。结果:本实验室结合前期工作的基础,得出与T细胞耗竭相关的几个因素是:抗酸染色、空洞情况、病程长短。以相关的几个因素将51例肺结核病人和16例健康志愿者分成4组,即健康对照组(HD组)、痰抗酸染色阴性肺结核病人组(N-TB组)、痰抗酸染色阳性的非空洞性肺结核病人组(PNC-TB组)以及痰抗酸染色阳性空洞性肺结核病人组(PC-TB组)。1,相对于健康对照组,痰抗酸染色阴性结核病人组及痰抗酸染色阳性的非空洞性结核病人组的T细胞增殖能力及分泌IFN-γ能力显著增高(P0.05);2,所有肺结核病人组的IL-2表达水平较健康对照组明显下降(P0.05),但肺结核病人各组之间无显著性差异(P0.05);3,与上述两组相比,痰抗酸染色阳性空洞性肺结核病人组T细胞增殖能力和分泌IFN-γ能力显著降低(P0.05);4,所有肺结核病人组TIM-3的表达水平较健康对照组显著增高(P0.05),但在肺结核病人组之间的差异不明显(P0.05);5,痰抗酸染色阳性空洞型肺结核人CD4~+、CD4~+TIM-3~+T细胞的数目有所下降,但差异尚不显著(P0.05),可能与样本量较小有关;6,痰抗酸染色阳性空洞型肺结核病人血小板数目较其他各组出现减少,差异有统计学意义(P0.05),各组病人外周血中红细胞、血红蛋白、白细胞以及单个核细胞之间无显著性差异。结论:1,TIM-3可能作用于肺结核病发展的整个过程;2,T细胞分泌IL-2的能力在肺结核发展的早期就开始下降;3,肺结核病人发生T细胞耗竭与抗酸染色、空洞情况、病程长短几个因素相关;4,本研究证实持续痰菌阳性的空洞性肺结核病人T细胞功能出现耗竭,提示人体内大量结核分枝杆菌持续感染可导致T细胞功能耗竭。结核病是由结核分枝杆菌引起的一种慢性传染病,它不仅可以引起肺结核也可以肺外结核,结核分枝杆菌在宿主体内长期持续存在,损害宿主的免疫功能。类似于HBV、HCV、HIV等病毒性疾病持续感染所致的T细胞耗竭,结核分枝杆菌持续感染所致的T细胞功能耗竭越来越受到重视。发生耗竭的T细胞主要表现是细胞表面抑制性受体增加,细胞增殖能力下降,细胞毒作用丧失以及细胞因子分泌能力降低。结核病患者一旦发生T细胞耗竭,机体表现为免疫功能低下,疾病持续恶化,很难恢复。为逆转结核病T细胞耗竭,恢复机体免疫功能,本研究构建动物模型来验证两种逆转T细胞耗竭药物的作用。目的:研究结核分枝杆菌抗原持续刺激致T细胞功能耗竭的机制,探究补肾健脾方和二甲双胍逆转T细胞功能耗竭的作用。方法:将3周龄的实验动物随机分为5组:正常对照组、抗原短暂免疫组、免疫耗竭未治疗组、免疫耗竭补肾健脾解毒方组、免疫耗竭二甲双胍治疗组。实验第1周利用BCG初免,PPD(结核菌素纯蛋白衍化物蛋白)持续刺激C57BL/6小鼠,模拟结核分枝杆菌抗菌抗原在病人体内持续存在所造成慢性感染的过程,构建T细胞功能耗竭模型。从第7周开始,给予免疫耗竭二甲双胍治疗组饮水中给药治疗至第26周,从第24周开始,给予补肾健脾解毒方组小鼠灌胃治疗至第26周。治疗完成后检测各组小鼠细胞免疫水平,包括利用流式细胞术检测脾脏CD4~+和CD8~+T细胞PD-1的表达水平、CD4~+和CD8~+T细胞的数目;利用ELISA检测脾脏淋巴细胞分泌IL-2和IFN-γ的水平;利用肺脏BCG攻毒实验检测抗感染能力;利用流式细胞术检测造血干细胞占小鼠骨髓有核细胞的比例。结果:与抗原短暂刺激组相比,免疫耗竭未治疗组CD4~+和CD8~+T细胞数目显著下降(P0.05),细胞表面PD-1的表达水平显著增高,差异有统计学意义(P0.01);脾淋巴细胞分泌细胞因子IL-2、IFN-γ的水平明显降低,差异有统计学意义(P0.01);小鼠抗感染的能力显著下降,差异有统计学意义(P0.01);说明PPD抗原持续刺激致T细胞耗竭模型构建成功。经补肾健脾方治疗后,小鼠脾脏CD4~+T细胞表面PD-1的表达水平显著降低,差异有统计学意义(P0.01),CD4~+和CD8~+T细胞数目得到明显恢复(P0.05);小鼠脾脏T淋巴细胞分泌IL-2、IFN-γ的水平明显增高,差异有统计学意义(P0.01);小鼠抗BCG感染的能力显著增强,差异有统计学意义(P0.01);小鼠脾脏的炎症反应明显减轻,差异有统计学意义(P0.05)。经二甲双胍干预后,脾CD4~+T细胞PD-1的表达显著性降低,差异有统计学意义(P0.01);脾淋巴细胞分泌IL-2和IFN-γ的水平明显增高,差异有统计学意义(P0.01);小鼠抗感染的能力显著性增强,差异有统计学意义(P0.01)。结论:1,抗原PPD持续刺激可致T细胞发生耗竭;2,补肾健脾方能逆转抗原持续刺激导致的小鼠T细胞功能耗竭,可能为临床治疗结核病T细胞功能耗竭提供参考;3,二甲双胍对T细胞功能耗竭的进展有干预作用,提示患有二型糖尿病或者空腹血糖受损的肺结核病人口服二甲双胍的必要性。
[Abstract]:Worldwide, the prevalence and fatality of tuberculosis are very high, especially in developing countries such as China and India. Tuberculosis is a heavy burden to society and individuals. In the early stages of Mycobacterium tuberculosis, most people can prevent further development of tuberculosis through their own immunity, and about 10% of the population end up. It is developed into active tuberculosis. In the late stage of Mycobacterium tuberculosis (Mtb) infection, the immune system caused the recovery of Mycobacterium tuberculosis and the formation of chronic infectious tuberculosis. The main manifestations of the body are low immune function under the effect of chronic chronic infection. Why are the patients with severe pulmonary tuberculosis manifested as low immunity? In this study, the mechanism of the depletion of T cells in the patients with chronic pulmonary tuberculosis was investigated by analyzing several functional indexes related to the depletion of the function of T cells in the peripheral blood of the patients with clinical pulmonary tuberculosis. The purpose of this study was to analyze the causes of the low immune function of the patients with chronic pulmonary tuberculosis and to explore the holding of Mycobacterium tuberculosis in the human body. Whether continuous infection causes T cell function depletion and study the changes of T cell function in patients with persistent phlegm positive cavitary pulmonary tuberculosis. Methods: 51 cases of pulmonary tuberculosis patients in Lanzhou lung hospital and 16 healthy volunteers were selected, and the peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs).1 were isolated and the enzyme was used. The secretory level of IFN- gamma in peripheral blood T lymphocytes stimulated by antigen ESAT-6 and CFP-10 was detected by ELISPOT. 2, the secretion level of IL-2 in the supernatant of T lymphocyte culture was detected by ELISA technique; 3, PBMCs was cultured under the stimulation of Purified Protein Derivative of Tuberculin (PPD), and the increase of T cells in peripheral blood was analyzed by flow cytometry. Colonization; 4, the number of CD4~+, CD8~+T cells, and the expression of T cell immunoglobulin 3 (T cell immunoglobulin mucin domain containingmolecules 3, TIM-3), and the expression of CD4~+, CD8~+T cell surface immunosuppressive related molecules; 5, the blood hemogram of the same period of the same period of the patients, the red and blood eggs of the peripheral blood were analyzed. White, white blood cells, platelets and mononuclear cells; 6, by analyzing the relationship between sex, age, course of disease, initial (complex) treatment, sputum acid staining, lung imaging findings and T cell depletion related indicators to explore the depletion of T cells in several types of chronic tuberculosis patients. Results: this laboratory combined with previous work. Several factors related to the depletion of T cells were: acid stain, cavity and length of disease. 51 cases of tuberculosis patients and 16 healthy volunteers were divided into 4 groups, that is, healthy control group (group HD), sputum acid negative staining negative pulmonary tuberculosis group (group N-TB), phlegm positive non cavity pulmonary tuberculosis The group (group PNC-TB) and the sputum acid positive cavitary pulmonary tuberculosis group (group PC-TB).1, compared with the healthy control group, the T cell proliferation ability and the secretion of IFN- gamma in the group of phlegm negative acid negative tuberculosis patients and phlegm positive non cavity tuberculosis patients were significantly increased (P0.05); 2, IL-2 table of all pulmonary tuberculosis patients group. There was a significant decrease in the level of reaching the level of the healthy control group (P0.05), but there was no significant difference between the pulmonary tuberculosis patients (P0.05). 3, compared with the two groups, the T cell proliferation ability and the secretion of IFN- gamma in the phlegm positive cavitary pulmonary tuberculosis patients were significantly lower (P0.05); 4, the expression level of TIM-3 in all patients with tuberculosis was more than that of the healthy control group. There was a significant increase (P0.05), but the difference between the patients with pulmonary tuberculosis was not significant (P0.05); 5, the number of CD4~+ and CD4~+TIM-3~+T cells in the phlegm positive cavitary pulmonary tuberculosis decreased, but the difference was not significant (P0.05), which may be related to the small sample size; 6, the number of platelets in the phlegm positive cavitary pulmonary tuberculosis patients was more than that of the others. There was no significant difference in each group (P0.05). There was no significant difference between the peripheral blood red blood cells, hemoglobin, leukocyte and mononuclear cells in the peripheral blood of each group. Conclusion: 1, TIM-3 may act on the whole process of pulmonary tuberculosis development; 2, the ability of T cells to secrete IL-2 begins to decline in the early stage of tuberculosis development; 3, tuberculosis. T cell depletion was associated with a number of factors associated with acid resistance, cavity conditions, and duration of the disease. 4, this study confirmed the depletion of T cell function in patients with persistent phlegm positive cavitary tuberculosis, suggesting that the persistent infection of a large number of Mycobacterium tuberculosis in the human body can lead to the exhaustion of T cells. Tuberculosis is caused by Mycobacterium tuberculosis. Chronic infectious diseases can not only cause tuberculosis but also extrapulmonary tuberculosis. Mycobacterium tuberculosis persists in the host for a long time and damages the immune function of the host. It is similar to the depletion of T cells caused by persistent infection of viral diseases such as HBV, HCV, HIV, and the depletion of T cells caused by the persistent infection of Mycobacterium tuberculosis. The main manifestations of the depleted T cells are the increase of the cell surface inhibitory receptor, the decline of cell proliferation, the loss of cytotoxic effect and the decrease of cytokine secretion ability. Once the patients with tuberculosis occur T cells exhaustion, the body is immune to low immune function, and the disease continues to deteriorate, it is difficult to restore the exhaustion of tuberculosis T cells. To restore the immune function of the body, this study constructs an animal model to verify the effect of two kinds of reverse T cell exhaustion drugs. Objective: To study the mechanism of T cell depletion by the continuous stimulation of Mycobacterium tuberculosis, and to explore the role of the kidney invigorating spleen recipe and metformin in reversing the function depletion of T cells. Methods: the experimental animals of 3 weeks of age were randomly divided into two groups. The 5 groups: the normal control group, the antigen transient immune group, the immune exhaustion untreated group, the immune exhausted invigorating kidney invigorating spleen and the detoxification group, the immune exhaustion metformin treatment group. The experiment first weeks using BCG primordial immunity and PPD (tuberculin pure protein derivative protein) continuously stimulated C57BL/6 mice, and simulated the persistent presence of Mycobacterium tuberculosis antigenic antigen in the patient's body. In the process of chronic infection, the function depletion model of T cells was constructed. From seventh weeks, the treatment group of metformin in the treatment group of the immune depleted metformin treatment group was treated for twenty-sixth weeks. From the twenty-fourth week, the mice were given the kidney invigorating spleen and the spleen detoxification group for twenty-sixth weeks. After the treatment was completed, the cell immune level of each group was detected, including the use of flow. The expression level of PD-1, the number of CD4~+ and CD8~+T cells, the number of CD4~+ and CD8~+T cells, the level of the secretion of IL-2 and IFN- in the spleen lymphocytes were detected by ELISA, and the anti infective ability of the spleen lymphocytes to detect the secretion of IL-2 and IFN- gamma; the detection of anti infection ability by the lung BCG attack test; the ratio of hematopoietic stem cells to the marrow nucleated cells in mice was detected by flow cytometry. Compared with the primary transient stimulation group, the number of CD4~+ and CD8~+T cells in the untreated group was significantly decreased (P0.05), and the expression level of PD-1 on the cell surface was significantly higher, the difference was statistically significant (P0.01); the level of cell factor IL-2, IFN- gamma decreased significantly (P0.01), and the ability of anti infection in mice was significant (P0.01). The difference was statistically significant (P0.01), indicating that the T cell depletion model was constructed successfully by the continuous stimulation of PPD antigen. After the treatment of kidney invigorating spleen, the expression level of PD-1 on the surface of CD4~+T cells in spleen of mice decreased significantly (P0.01), the number of CD4~+ and CD8~+T cells recovered significantly (P0.05), and the T lymph nodes of mouse spleen were fine. The level of IL-2 and IFN- gamma increased significantly, the difference was statistically significant (P0.01), the ability of mice to resist BCG infection was significantly enhanced, the difference was statistically significant (P0.01), the inflammatory response of spleen in mice was significantly reduced, and the difference was statistically significant (P0.05). The expression of PD-1 in spleen CD4~+T cells decreased significantly after two metformin, and the difference had a significant difference. Study significance (P0.01); the secretion of IL-2 and IFN- gamma in spleen lymphocytes increased significantly, the difference was statistically significant (P0.01); the ability of anti infection in mice increased significantly (P0.01). Conclusion: 1, the continuous stimulation of antigen PPD can cause depletion of T cells; 2, the recipe for invigorating the kidney and invigorating the spleen can reverse the T fines caused by the continuous antigen stimulation of the antigen. The depletion of cell function may provide a reference for the clinical treatment of T cell depletion of TB cells; 3, metformin has an intervention in the progress of T cell depletion, suggesting the necessity of oral metformin in patients with type two diabetes or impaired fasting blood glucose.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R521;R-332

【相似文献】