肺炎支原体感染诱导鼠脾淋巴细胞凋亡的研究

发布时间：2018-03-09 04:35

本文选题：肺炎支原体　切入点：鼠脾淋巴细胞　出处：《中国医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 前言肺炎支原体(Mycoplasma pneumoniae,Mp)感染可引起原发性非典型肺炎、咽炎、气管炎、支气管炎等呼吸道疾病,严重者可引起复杂的肺外合并症和致死性肺炎。其感染不仅发病率较高,且病程长,易复发。肺炎支原体感染的发病机理至今并未清楚,主要观点趋向于肺炎支原体直接侵入呼吸道上皮细胞引起炎症反应和感染导致免疫紊乱反应两种学说。近几年来,有不少研究者通过对数例临床支原体肺炎患儿外周血检测,发现患儿存在外周血淋巴细胞凋亡增强现象,且外周血淋巴细胞凋亡率与CD4~+细胞百分率和CD4~+/CD8~+细胞比值呈明显负相关。研究报道外周血成熟淋巴细胞凋亡加速可能是引起该组患者免疫功能降低、病程长、易复发的一个重要原因。那么,Mp能否体外诱导淋巴细胞凋亡,Mp感染与CD4~+T淋巴细胞凋亡的关系如何,国内外尚未见报道。本实验通过用不同浓度的Mp感染体外培养的小鼠脾淋巴细胞,用PE-Cy5 Rat anti-Mouse CD4标记CD4~+T淋巴细胞,采用AO/EB荧光染色技术和Annexin-V FITC/PI流式细胞仪技术检测总淋巴细胞和CD4~+T淋巴细胞的调亡情况,对Mp诱导淋巴细胞凋亡进行探讨,为进一步阐明肺炎支原体感染的发病机理奠定基础。实验方法一、肺炎支原体的培养将本实验室保存的肺炎支原体菌株复苏后接种于支原体液体培养基中进行培养,显微镜下观察到有支原体生长即作传代培养。二、淋巴细胞获取选用体重为20克左右的昆明小鼠,无菌取脾,用塑料针芯轻轻挤压,磨碎脾脏,收集细胞悬液,离心弃上清,加入红细胞裂解液裂解红细胞,PBS洗两次,用RPMI-1640培养基将细胞浓度调节至1×10~6/ml。三、凋亡的诱导取对数生长期的肺炎支原体菌液,高速离心获取菌体沉淀,PBS洗两次,用紫外线分光光度法进行Mp蛋白定量来确定Mp浓度。将脾淋巴细胞接种在24孔组织培养板上,分别加入不同浓度(50μg/ml,100μg/ml,200μg/ml和300μg/ml)的Mp悬液,同时设正常细胞对照组,于37.5℃、5%CO_2饱和湿度条件下培养4小时。四、凋亡的检测 1、荧光显微镜观察细胞凋亡的形态学改变。 2、用AO/EB荧光染色技术和和Annexin-V FITC/PI双染流式细胞仪技术检测分析总淋巴细胞凋亡率。 3、用PE-Cy5 Rat anti-Mouse CD4标记CD4~+T淋巴细胞,Annexin-V FITC/PI流式细胞仪检测CD4~+T淋巴细胞的调亡情况。 4、所有资料用(?)±s表示,各组间采用SPSS软件进行方差分析。对照组与各实验组进行比较,各实验组之间再进行比较。实验结果 1、不同剂量Mp体外诱导鼠脾淋巴细胞4h后,经AO/EB染色在荧光显微镜下可见大量桔黄色的凋亡细胞,这些细胞呈典型的凋亡形态学改变:染色质浓缩,包膜起泡,出芽,细胞核碎裂成点状,被染成大小不一、致密浓染的黄绿色颗粒。 2、AO/EB荧光染色及流式细胞仪二种方法检测Mp诱导组总淋巴细胞凋亡率及CD4~+T淋巴细胞调亡百分率明显高于正常对照组(p＜0.01),且两种方法检测凋亡百分率结果一致。 3、总淋巴细胞及CD4~+T淋巴细胞凋亡率随Mp感染剂量的增加而增加。结论 1、Mp在体外能诱导鼠脾淋巴细胞出现明显凋亡。 2、随着Mp体外感染剂量的增加,鼠脾淋巴细胞及CD4~+T淋巴细胞凋亡率也越来越高,表明Mp以剂量依赖方式体外诱导鼠脾淋巴细胞和CD4~+T淋巴细胞凋亡。
[Abstract]:Preface
Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) infection can cause primary atypical pneumonia, pharyngitis, tracheitis, bronchitis and other respiratory diseases, severe cases can cause complex pulmonary complications and deaths from pneumonia. The infection is not only a higher incidence, and the course of disease is long, easy to relapse. The pathogenesis of Mycoplasma pneumoniae infection so far not clear, main ideas tend to Mycoplasma pneumoniae directly invade the airway epithelial cells and cause inflammation and infection causes immune disorder reaction of two kinds of theories. In recent years, many researchers through the log cases of mycoplasma pneumonia in peripheral blood were detected, found in patients with peripheral blood lymphocyte apoptosis enhancement, and peripheral blood the lymphocyte apoptosis rate and the percentage of CD4~+ cells and CD4~+/CD8~+ cell ratio was negatively correlated. Reports on peripheral blood lymphocyte maturation accelerated apoptosis may be caused by this Patients with decreased immune function, long course of disease, an important cause of recurrence. So, Mp can induce apoptosis of lymphocyte in vitro, the relationship between Mp infection and CD4~+T lymphocyte apoptosis, have been reported at home and abroad. Through the experiments with different concentrations of Mp infected mice spleen lymphocytes in vitro, using PE-Cy5 Rat anti-Mouse CD4 mark CD4~+T lymphocytes, using AO/EB fluorescence staining and Annexin-V FITC/PI flow cytometry technique and CD4~+T lymphocyte apoptosis, to investigate Mp induced lymphocyte apoptosis, lay the foundation for further understanding the pathogenesis of Mycoplasma pneumoniae infection.
Experimental method
One, the culture of Mycoplasma pneumoniae
The Mycoplasma pneumoniae strain was resuscitation and inoculated in the liquid culture medium of Mycoplasma. The growth of Mycoplasma was observed under microscope.
Two, lymphocyte acquisition
A Kunming mouse with a body weight of 20 grams was selected. The spleen was taken aseptic, gently squeezed with plastic core, and the spleen was grinded. The cell suspension was collected. Centrifugation was used to dissolve the supernatant. The erythrocyte lysate was split into red blood cells, washed with PBS for two times, and the cell concentration was adjusted to 1 * 10~6/ ml. with RPMI-1640 medium.
Three, the induction of apoptosis
Mycoplasma pneumoniae bacteria in logarithmic growth phase, high speed centrifugation was precipitated, washed two times with PBS, Mp protein assay was performed to determine the Mp concentration by ultraviolet spectrophotometry. Spleen cells were seeded in 24 well tissue culture plates, respectively with different concentrations (50 g/ml, 100 g /ml, 200 g/ml and 300 g/ml) Mp suspension, and the normal cell control group, 5%CO_2 at 37.5 DEG C, saturated humidity conditions and cultured for 4 hours.
Four, the detection of apoptosis
1, the morphological changes of cell apoptosis were observed by fluorescence microscope.
2, the total lymphocyte apoptosis rate was detected by AO/EB fluorescence staining and Annexin-V FITC/PI double dye flow cytometry.
3, CD4~+T lymphocytes were labeled with PE-Cy5 Rat anti-Mouse CD4 and Annexin-V FITC/PI flow cytometry was used to detect the apoptosis of CD4~+T lymphocytes.
4, all the data were expressed with (?) + s, and the SPSS software was used to analyze the variance between the groups. The control group was compared with the experimental groups, and the experimental groups were compared again.
experimental result
1, mouse spleen cells 4H induced by different doses of Mp in vitro, the under fluorescent microscope showed a large amount of orange AO/EB staining of apoptotic cells, these cells showed typical apoptosis morphological changes: chromatin condensation, membrane blistering, budding, punctate nuclear fragmentationinto, dyed into different sizes, densely stained green and yellow particles.
2, AO/EB fluorescence staining and flow cytometry detected the total lymphocyte apoptosis rate and CD4~+T lymphocyte apoptosis percentage in Mp induced group by two methods. The percentage of CD4~+T lymphocyte apoptosis was significantly higher than that in the normal control group (P < 0.01), and the percentage of apoptosis detected by the two methods was the same.
3, the apoptosis rate of total lymphocyte and CD4~+T lymphocyte increased with the increase of Mp infection dose.
conclusion
1, Mp can induce obvious apoptosis in rat spleen lymphocyte in vitro.
2, with the increase of Mp infection in vitro, the apoptosis rate of rat splenic lymphocytes and CD4~+T lymphocytes is also increasing. It indicates that Mp induces apoptosis of rat splenic lymphocytes and CD4~+T lymphocytes in a dose-dependent manner.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R375;R392

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