T淋巴细胞相关协同刺激分子在重症肺炎发病机制中的作用研究

发布时间：2018-04-02 14:00

本文选题：重症肺炎　切入点：免疫无能　出处：《广州医学院》2009年硕士论文

【摘要】： 背景重症肺炎目前具体的发病机制不详。研究发现重症肺炎患者淋巴细胞显著减少,并与患者的预后密切相关。是什么导致重症肺炎淋巴细胞减少?研究表明重症肺炎患者外周血中协同刺激分子表达异常,可能导致T淋巴细胞处于“免疫无能(anergy)”状态。其中是因为T淋巴细胞相关的协同刺激分子CD28,CD152及CD86等的表达异常,还是因为CD28/CD152这对相互拮抗的信号分子的比例异常导致了T淋巴细胞处于“免疫无能”状态?目前尚不清楚。研究发现胸腺肽-α1(Tα1)在机体免疫应答过程中起着重要的作用,并认为其可能是通过增强T淋巴细胞功能而调节免疫系统的。研究观察到胸腺肽a1可以改善脓毒性休克患者的T淋巴细胞增殖,调节机体的细胞免疫功能。可见,胸腺肽a1有调节机体免疫异常的功能,是否通过调控协同刺激信号来影响T淋巴细胞的增殖?尚待进一步的研究。目的在蛋白水平上研究重症肺炎T细胞相关协同刺激分子与其细胞增殖能力的关系,以探讨该病的发病机制;在此基础上,观察胸腺肽-α1对重症肺炎T淋巴细胞协同刺激分子及其细胞增殖能力的影响,并进一步探讨T淋巴细胞协同刺激分子在重症肺炎免疫异常中的可能作用。资料和方法 1标本来源 2007年8月一2008年06月入住广州市第一人民医院呼吸科的重症肺炎患者,均符合美国胸科学会(ATS)于2007年制定的重症肺炎诊断标准。健康对照组来自同期门诊健康体检者。经本人及家属知情同意后抽取外周静脉血。重症肺炎患者21例,抽血2mL,EDTA-K3抗凝;其中11例多抽血13mL,肝素抗凝;另外其中11例于住院治疗后10天再次抽血2mL(其中28天患者预后为8例好转、3例死亡)。健康人12名抽血2mL,EDTA-K3抗凝;其中10名多抽血8mL肝素抗凝。 2方法 2.1 T淋巴细胞相关协同刺激分子的检测:取EDTA-K3抗凝血标本。设测定管及同型对照管。检测T淋巴细胞上的四个表位(CD4, CD8, CD28, CD152)及相关的单核细胞的两个表位(CD86,HLA-DR)。用FITC、PE、PC5、APC和APCcy7荧光素直接标记单克隆抗体。检测前校准流式细胞仪的光路和液路。将光路调整至最佳,检测阴性对照,调整电压和颜色补偿。利用FACScan及CELLQuest软件收集分析数据。使用前向角散射光侧向角散射光双参数设门。收集10000个细胞。以百分率表示每群细胞的表达。 2.2 T淋巴细胞的增殖试验 2.2.1人外周血单个核细胞(PBMC)提取:密度梯度离心法提取重症肺炎病人及健康人的外周血标本(PBMC)。立即使用或冻存,用前溶解。 2.2.2单个核细胞(PBMC)PKH26染色及培养:参考PKH26染料说明书对PBMC进行染色。重悬成1×106 / ml的浓度,接种于24孔培养板孔中。实验分为4组:①空白对照组PBS+PKH26染色后PBMC;②阳性对照组PHA+PKH26染色后PBMC;③胸腺肽-α1+内毒素+PKH26染色后PBMC;④内毒素PKH26+染色后PBMC。每孔终体积约0.6 ml浓度1×106 / ml,在37℃、5 %CO2条件下培养5天。 2.2.3 T淋巴细胞增殖的检测:取出培养5天后的PKH26染色细胞,分两管,每管0.3ml ,离心洗涤,加入荧光素标记的PC5—CD3 1μg/ 106细胞,混匀后室温暗处孵育,洗涤后,用2 g/L多聚甲醛固定,利用流式细胞仪检测及数据用CELLQuest软件和ModFitTM软件分析得到T淋巴细胞的增殖指数(PF)及增殖细胞的前体频率(PI)。 2.2.4体外培养T淋巴细胞及协同分子的检测:取未染色的单个核细胞,重悬成1×106 / ml的浓度,接种于24孔培养板孔中。实验分为4分组:①空白对照组PBS+未染色PBMC;②阳性对照组PHA+未染色PBMC;③胸腺肽-α1+内毒素+未染色PBMC;④内毒素+未染色PBMC。每孔终体积约0.6 ml,浓度1×106 / ml,在37℃、5 %CO 2条件下培养5天。取出培养5天后未染色的细胞,分两管,每管0.3ml ,离心洗涤,加入荧光素标记的FITC—CD28/PE—CD152/PC5—CD3各1μg/ 106细胞,混匀后室温暗处孵育,洗涤后,用2 g/L多聚甲醛固定,利用流式细胞仪检测及数据用CELLQuest软件分析协同刺激分子CD28、CD152的表达。 2.3统计学方法:所有实验数据均用SPSS 11.5软件分析,计量资料以均数士标准差(士S)表示,两组均数比较采用t检验。随机区组设计资料采用方差分析,用LSD法进行数据间的多重比较,取P0.05差异有统计学意义。结果 1重症肺炎T淋巴细胞、亚群及相关协同刺激分子:21例重症肺炎患者CD3、CD4、CD86、HLA-DR、CD28/ CD152、CD4/CD8较12例健康对照组减少,有统计学显著性差异(P 0.05);CD8、CD28,CD152表达较健康对照组增加,有统计学显著性差异(P 0.05)。 2 28天存活组重症肺炎患者治疗前后(10天)T淋巴细胞、亚群及相关协同刺激分子的变化:存活组8例重症肺炎入院第10天和第1天比较APACHEⅡ评分显著减少(P=0.000);CD28、CD152、CD86、HLA-DR的表达显著升高(P 0.05);CD3T细胞也显著增加(P=0.030);CD8CD3,CD4CD3阳性的T细胞无显著变化(P 0.05)。 3 PKH26标记重症肺炎患者外周血单个核细胞(PMBC)的情况: PKH26标记PMBC,在直方图上PKH26荧光强度出现明显右偏,达104,呈单峰,阳性率为96.48%。荧光显微观察PKH26标记后的PMBC,可见其细胞发红色荧光。 4重症肺炎患者外周血T淋巴细胞对抗原刺激的增殖能力:10例重症肺炎患者外周血T淋巴细胞对内毒素(LPS)刺激的增殖指数PI(1.55±0.42)与增殖细胞的前体频率PF(0.02±0.01)低于10例健康人组的PI (2.15±0.29)与PF(0.04±0.02),有统计学差异(P 0.05)。 5胸腺肽-α1对重症肺炎外周血T淋巴细胞增殖的影响 5.1光学显微镜观察胸腺肽-α1对重症肺炎外周血T淋巴细胞增殖的影响:不同干预因素组重症肺炎外周血T淋巴细胞在体外培养5天后,用显微镜观察(×10倍),可见PHA组及胸腺肽-α1加内毒素组出现明显的淋巴细胞母细胞化,其他两组未见明显的淋巴细胞母细胞化。 5.2胸腺肽-α1对重症肺炎外周血T淋巴细胞增殖的影响:10例重症肺炎PHA组、胸腺肽-α1加内毒素组及内毒素组T淋巴细胞的增殖细胞的前体频率(PF)分别为:0.03±0.03、0.03±0.02、0.02±0.01;T淋巴细胞的增殖指数(PI)分别为:1.68±0.49、1.84±0.53、1.55±0.42。胸腺肽-α1加内毒素组T淋巴细胞的增殖指数与增殖细胞的前体频率比内毒素组的高,有统计学显著性差异(P 0.05);胸腺肽-α1加内毒素组T淋巴细胞的增殖指数及增殖细胞的前体频率与PHA组的比较,无统计学显著性差异(P 0.05)。 6胸腺肽-α1对重症肺炎外周血T淋巴细胞协同刺激分子的影响 6.1胸腺肽-α1对重症肺炎外周血T淋巴细胞CD28协同刺激分子的影响: PBS组(67.40±6.45)%、PHA组(72.21±7.05)%、胸腺肽-α1加内毒素组(73.70±8.66)%及内毒素组(66.94±11.85) %。胸腺肽-α1加内毒素组T淋巴细胞CD28协同刺激分子的表达比内毒素组及PBS组的高,有统计学显著性差异(P 0.05);胸腺肽-α1加内毒素组T淋巴细胞CD28协同刺激分子的表达与PHA组的比较,无统计学显著性差异(P=0.479)。 6.2胸腺肽-α1对重症肺炎外周血T淋巴细胞CD152协同刺激分子的影响: PBS组(3.01±1.08)%、PHA组(2.69±1.23)%、胸腺肽-α1加内毒素组(2.41±0.77)%及内毒素组(2.86±1.03) %。胸腺肽-α1加内毒素组T淋巴细胞CD152协同刺激分子的表达比内毒素组及PBS组的低,有统计学显著性差异(P 0.05);胸腺肽-α1加内毒素组T淋巴细胞CD152协同刺激分子的表达与PHA组的比较,无统计学显著性差异(P=0.306)。 6.3胸腺肽-α1对重症肺炎外周血T淋巴细胞协同刺激分子CD28/CD152比值的影响: PBS组(25.26±9.57)、PHA组(31.38±11.67)、胸腺肽-α1加内毒素组(33.30±9.96)及内毒素组(21.13±5.39)。胸腺肽-α1加内毒素组T淋巴细胞协同刺激分子CD28/CD152的比值比内毒素组及PBS组的大,有统计学显著性差异(P 0.05);胸腺肽-α1加内毒素组T淋巴细胞协同刺激分子CD28/CD152的比值与PHA组的比较,无统计学显著性差异(P=0.490)。结论 1.重症肺炎患者外周血T淋巴细胞存在“免疫无能”,导致了T淋巴细胞及CD4亚群减少,抑制了机体的免疫功能。 2. T淋巴细胞相关协同刺激分子CD28、CD152及CD86表达异常参与了重症肺炎发病的病理生理过程。 3.体外胸腺肽-α1可通过调节重症肺炎患者外周血T淋巴细胞协同刺激分子CD28、CD152表达;来改善其对抗原刺激的增殖能力。
[Abstract]:background
At present, the specific pathogenesis of severe pneumonia is unknown. The study found that lymphocytes of patients with severe pneumonia was significantly reduced, and is closely related to the prognosis of the patients. What caused the reduction of lymphocytes? Study showed that patients with severe pneumonia in the peripheral blood of abnormal expression of costimulatory molecules, may lead to T lymphocytes in immune incompetence (anergy) ". One is because the T lymphocyte associated costimulatory molecules CD28, expression of CD152 and CD86 anomalies, or because the CD28/CD152 of antagonistic signaling molecules leads to the abnormal rate of T lymph cells in immune incompetent"? Is unclear. The study found that thymosin alpha 1 (T alpha 1) plays an important the role during the immune response, and it may regulate the immune system by enhancing the function of T lymphocytes and the study observed. Thymosin A1 can improve The proliferation of T lymphocytes in patients with septic shock and the regulation of cellular immune function can be seen. Thymosin A1 has the function of regulating the immune abnormality. Whether we need to regulate the co stimulatory signal to affect the proliferation of T lymphocytes remains to be further studied.
objective
Study on the relationship between severe pneumonia T cells at the protein level associated costimulatory molecules and cell proliferation, to investigate the pathogenesis of the disease; on this basis, to observe the effect of thymosin alpha 1 on severe pneumonia in T lymphocytes and cell proliferation, and to further explore the T lymphocytes in immune severe pneumonia the possible role of abnormal.
Information and methods
1 source of specimen
In August 2007 2008 06 a month in the Department of respiration of Guangzhou No.1 People's Hospital of the patients with severe pneumonia, are in line with the American Thoracic Society (ATS) in diagnosis of severe pneumonia standard enacted in 2007. The healthy control group from healthy subjects served by themselves and their families. Informed consent from peripheral venous blood. 21 cases of patients with severe pneumonia, blood 2mL, EDTA-K3 anticoagulant of which 11 cases of multiple blood 13mL; heparin; in addition, of which 11 cases in the hospital 10 days after treatment were 2mL (28 days for the prognosis of patients with 8 cases improved, 3 cases died). 12 healthy people were 2mL, EDTA-K3 anticoagulation; including 10 more blood 8mL heparin.
2 method
2.1 the detection of T lymphocyte associated costimulatory molecules: take EDTA-K3 anticoagulant samples. Test tubes and control tube. The detection of T lymphocyte on the four epitopes (CD4, CD8, CD28, CD152) and mononuclear cells of two epitopes (CD86, HLA-DR). FITC, PE PC5, directly labeled monoclonal antibody APC and APCcy7 fluorescein. Before testing, calibration of flow cytometry light path and liquid. The light path adjustment to the best detection, negative control, voltage adjustment and color compensation. Using FACScan and CELLQuest software to analyze the data collected. Using forward scatter side scatter parameter gate. Collect 10000 cells expressed as a percentage of each group. The expression of the cell.
2.2 T lymphocyte proliferation test
2.2.1 human peripheral blood mononuclear cells (PBMC) were extracted: density gradient centrifugation was used to extract peripheral blood samples (PBMC) from severe pneumonia patients and healthy persons.
2.2.2 mononuclear cells (PBMC) staining and culture: PKH26 reference PKH26 dye staining for PBMC instructions. Resuspend into 1 * 106 / ml were inoculated into 24 well culture plate hole. The experiment was divided into 4 groups: blank control group PBS+PKH26 staining after PBMC; the positive control group were stained with PHA+PKH26 PBMC; the thymosin alpha 1+ endotoxin +PKH26 staining after PBMC PKH26+ staining after PBMC.; the endotoxin per hole final volume is about 0.6 ml the concentration of 1 * 106 / ml at 37 DEG C, cultured for 5 days under the condition of 5%CO2.
Detection of 2.2.3 T lymphocyte proliferation after 5 days of culture: remove PKH26 staining cells, divided into two tubes, each tube 0.3ml, centrifugal washing, adding FITC labeled PC5 - CD3 1 g/ 106 cells, mixing at room temperature after dark incubation, after washing, g/L fixed with 2 paraformaldehyde, analysis of T lymphocytes the proliferation index of CELLQuest and ModFitTM software by using flow cytometry and data (PF) precursor frequency and proliferation of cells (PI).
Detection of T lymphocyte 2.2.4 were cultured in vitro and CO molecules: the mononuclear cells were not stained, resuspended into 1 * 106 / mL concentration, were inoculated into 24 well culture plate. The experiment was divided into 4 groups: blank control group PBS+ without PBMC staining; the positive control group PHA+ without the PBMC staining; thymosin alpha 1+ endotoxin + unstained PBMC; the endotoxin + non staining of PBMC. per hole and final volume is about 0.6 ml, the concentration of 1 * 106 / ml at 37 DEG C, cultured for 5 days under the condition of%CO 2. 5 out of unstained cells cultured for 5 days, divided into two tubes, each tube 0.3ml, centrifugal washing. Adding a fluorescein labeled FITC - CD28/PE - CD152/PC5 - CD3 1 g/ 106 cells, mixing at room temperature after dark incubation, after washing, g/L fixed with 2 paraformaldehyde, using flow cytometry and data analysis using CELLQuest software, costimulatory molecules CD28, CD152 expression.
2.3 statistical methods: all data were analyzed by software SPSS 11.5, the measurement data to mean + standard deviation (S) said that the two groups were compared by t test. The random block design data using analysis of variance, multiple comparison between data with LSD method, P0.05 was statistically significant difference.
Result
1 severe pneumonia T lymphocyte subsets and costimulatory molecules: 21 cases of severe pneumonia in patients with CD3, CD4, CD86, HLA-DR, CD28/, CD152, CD4/CD8 compared with 12 cases of healthy control group, there was significant difference (P 0.05); CD8, CD28, CD152 expression increased compared with healthy control group, was statistically significant the difference (P 0.05).
The 228 day survival of patients with severe pneumonia before and after treatment (10 days) of T lymphocyte subsets, and costimulatory molecules: survival group 8 cases of severe pneumonia for tenth days and first days compared with APACHE score decreased significantly (P=0.000); CD28, CD152, CD86, HLA-DR expression was significantly increased (P 0.05); CD3T cells also increased significantly (P=0.030); CD8CD3, CD4CD3 + T cells showed no significant change (P 0.05).
3 PKH26 labeled peripheral blood mononuclear cells (PMBC) in severe pneumonia patients: PKH26 labeled PMBC, and PKH26 fluorescence intensity on the histogram showed a significant right deviation, reaching 104, showing a single peak. The positive rate was 96.48%. after fluorescence microscopy to observe PMBC after PKH26 labeling, and its cells showed red fluorescence.
T lymphocytes in peripheral blood of 4 patients with severe pneumonia on the antigen stimulated proliferation ability: 10 cases of severe pneumonia in patients with peripheral blood T lymphocyte proliferation index (LPS) on endotoxin stimulated PI (1.55 + 0.42) precursor frequency of PF and the proliferation of cells (0.02 + 0.01) is lower than that of 10 healthy people (2.15 of the PI group + 0.29) and PF (0.04 + 0.02), there was significant difference (P 0.05).
Effect of 5 thymosin - alpha 1 on the proliferation of T lymphocyte in peripheral blood of severe pneumonia
Effects of the proliferation of T lymphocytes in peripheral blood of 5.1 optical microscope observation of thymosin alpha 1 on severe pneumonia: T lymphocyte factor group of severe pneumonia in peripheral blood of different intervention after 5 days of culture in vitro, observed by microscope (x 10), visible PHA group and Thymosin alpha 1 plus endotoxin group lymphocyte blastisation obvious the other two groups, no obvious lymphocyte blastogenesis.
On the proliferation of T lymphocytes in peripheral blood of 5.2 of thymosin alpha 1 on severe pneumonia: 10 cases of severe pneumonia in the PHA group, the precursor frequency of thymosin alpha 1 plus endotoxin group and endotoxin group cell proliferation of T cells (PF) were: 0.03 + 0.03,0.03 + 0.02,0.02 + 0.01; T lymphocyte proliferation index (PI) respectively. The proliferation index of cell proliferation and 1.68 + 0.49,1.84 + 0.53,1.55 + 0.42. of thymosin alpha 1 plus endotoxin group T lymphocyte precursor frequency Binet toxin group, a statistically significant difference (P 0.05); compared with the precursor frequency of PHA cells proliferation index and proliferation of thymosin alpha 1 and the toxins of T lymphocytes, there was no statistically significant difference (P 0.05).
Effect of 6 thymosin - alpha 1 on the synergistic stimulator of T lymphocyte in peripheral blood of severe pneumonia
Effect of T CD28 lymphocytes in peripheral blood of 6.1 of thymosin alpha 1 on severe pneumonia and costimulatory molecules: PBS group (67.40 + 6.45)%, group PHA (72.21 + 7.05)%, thymosin alpha 1 plus endotoxin group (73.70 + 8.66)% and endotoxin group (66.94 + 11.85)% of thymosin alpha 1 with CD28 toxin group T lymphocyte costimulatory molecule expression of Binet toxin group and PBS group, there was significant difference (P 0.05); compared with PHA group the expression of thymosin alpha 1 plus endotoxin group T CD28 lymphocyte costimulatory molecules, there was no statistically significant difference (P=0.479).
Effect of T CD152 lymphocytes in peripheral blood of 6.2 of thymosin alpha 1 on severe pneumonia and costimulatory molecules: PBS group (3.01 + 1.08)%, group PHA (2.69 + 1.23)%, thymosin alpha 1 plus endotoxin group (2.41 + 0.77)% and endotoxin group (2.86 + 1.03)% of thymosin alpha 1 internal toxin group T lymph cell CD152 costimulatory molecule expression Binet toxin group and PBS group, there was significant difference (P 0.05); compared with PHA group the expression of thymosin alpha 1 plus endotoxin group T CD152 lymphocyte costimulatory molecules, there was no statistically significant difference (P=0.306).
Co stimulation T lymphocytes in peripheral blood of 6.3 of thymosin alpha 1 on severe pneumonia and effect of molecular ratio of CD28/CD152: PBS group (25.26 + 9.57), PHA group (31.38 + 11.67), thymosin alpha 1 plus endotoxin group (33.30 + 9.96) and endotoxin group (21.13 + 5.39) - alpha thymosin plus 1 LPS. In group of T lymphocytes and the ratio of CD28/CD152 Binet toxin group and PBS group, there was significant difference (P 0.05); thymosin alpha 1 plus endotoxin group T lymphocyte costimulatory molecules compared with PHA group the ratio of CD28/CD152, no statistically significant difference (P=0.490).
conclusion
1. the T lymphocyte in peripheral blood of patients with severe pneumonia has "immune inability", which leads to the decrease of T lymphocyte and CD4 subgroup, which inhibits the immune function of the body.
The abnormal expression of 2. T lymphocyte related co stimulators, CD28, CD152 and CD86, is involved in the pathophysiological process of severe pneumonia.
3. in vitro thymosin alpha 1 can improve the proliferation of T lymphocytes in patients with severe pneumonia by improving the expression of CO stimulatory molecules CD28 and CD152 in severe pneumonia patients.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】