本文選題：肺炎支原體　切入點：鼠脾淋巴細胞　出處：《中國醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： 前言 肺炎支原體(Mycoplasma pneumoniae,Mp)感染可引起原發(fā)性非典型肺炎、咽炎、氣管炎、支氣管炎等呼吸道疾病,嚴重者可引起復(fù)雜的肺外合并癥和致死性肺炎。其感染不僅發(fā)病率較高,且病程長,易復(fù)發(fā)。肺炎支原體感染的發(fā)病機理至今并未清楚,主要觀點趨向于肺炎支原體直接侵入呼吸道上皮細胞引起炎癥反應(yīng)和感染導(dǎo)致免疫紊亂反應(yīng)兩種學說。近幾年來,有不少研究者通過對數(shù)例臨床支原體肺炎患兒外周血檢測,發(fā)現(xiàn)患兒存在外周血淋巴細胞凋亡增強現(xiàn)象,且外周血淋巴細胞凋亡率與CD4~+細胞百分率和CD4~+/CD8~+細胞比值呈明顯負相關(guān)。研究報道外周血成熟淋巴細胞凋亡加速可能是引起該組患者免疫功能降低、病程長、易復(fù)發(fā)的一個重要原因。那么,Mp能否體外誘導(dǎo)淋巴細胞凋亡,Mp感染與CD4~+T淋巴細胞凋亡的關(guān)系如何,國內(nèi)外尚未見報道。本實驗通過用不同濃度的Mp感染體外培養(yǎng)的小鼠脾淋巴細胞,用PE-Cy5 Rat anti-Mouse CD4標記CD4~+T淋巴細胞,采用AO/EB熒光染色技術(shù)和Annexin-V FITC/PI流式細胞儀技術(shù)檢測總淋巴細胞和CD4~+T淋巴細胞的調(diào)亡情況,對Mp誘導(dǎo)淋巴細胞凋亡進行探討,為進一步闡明肺炎支原體感染的發(fā)病機理奠定基礎(chǔ)。 實驗方法 一、肺炎支原體的培養(yǎng) 將本實驗室保存的肺炎支原體菌株復(fù)蘇后接種于支原體液體培養(yǎng)基中進行培養(yǎng),顯微鏡下觀察到有支原體生長即作傳代培養(yǎng)。 二、淋巴細胞獲取 選用體重為20克左右的昆明小鼠,無菌取脾,用塑料針芯輕輕擠壓,磨碎脾臟,收集細胞懸液,離心棄上清,加入紅細胞裂解液裂解紅細胞,PBS洗兩次,用RPMI-1640培養(yǎng)基將細胞濃度調(diào)節(jié)至1×10~6/ml。 三、凋亡的誘導(dǎo) 取對數(shù)生長期的肺炎支原體菌液,高速離心獲取菌體沉淀,PBS洗兩次,用紫外線分光光度法進行Mp蛋白定量來確定Mp濃度。將脾淋巴細胞接種在24孔組織培養(yǎng)板上,分別加入不同濃度(50μg/ml,100μg/ml,200μg/ml和300μg/ml)的Mp懸液,同時設(shè)正常細胞對照組,于37.5℃、5%CO_2飽和濕度條件下培養(yǎng)4小時。 四、凋亡的檢測 1、熒光顯微鏡觀察細胞凋亡的形態(tài)學改變。 2、用AO/EB熒光染色技術(shù)和和Annexin-V FITC/PI雙染流式細胞儀技術(shù)檢測分析總淋巴細胞凋亡率。 3、用PE-Cy5 Rat anti-Mouse CD4標記CD4~+T淋巴細胞,Annexin-V FITC/PI流式細胞儀檢測CD4~+T淋巴細胞的調(diào)亡情況。 4、所有資料用(?)±s表示,各組間采用SPSS軟件進行方差分析。對照組與各實驗組進行比較,各實驗組之間再進行比較。 實驗結(jié)果 1、不同劑量Mp體外誘導(dǎo)鼠脾淋巴細胞4h后,經(jīng)AO/EB染色在熒光顯微鏡下可見大量桔黃色的凋亡細胞,這些細胞呈典型的凋亡形態(tài)學改變:染色質(zhì)濃縮,包膜起泡,出芽,細胞核碎裂成點狀,被染成大小不一、致密濃染的黃綠色顆粒。 2、AO/EB熒光染色及流式細胞儀二種方法檢測Mp誘導(dǎo)組總淋巴細胞凋亡率及CD4~+T淋巴細胞調(diào)亡百分率明顯高于正常對照組(p＜0.01),且兩種方法檢測凋亡百分率結(jié)果一致。 3、總淋巴細胞及CD4~+T淋巴細胞凋亡率隨Mp感染劑量的增加而增加。 結(jié)論 1、Mp在體外能誘導(dǎo)鼠脾淋巴細胞出現(xiàn)明顯凋亡。 2、隨著Mp體外感染劑量的增加,鼠脾淋巴細胞及CD4~+T淋巴細胞凋亡率也越來越高,表明Mp以劑量依賴方式體外誘導(dǎo)鼠脾淋巴細胞和CD4~+T淋巴細胞凋亡。
[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.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R375;R392

【參考文獻】

相關(guān)期刊論文 前10條

1 蔣玉紅,張忠國,王成;肺炎支原體肺炎患者外周血淋巴細胞凋亡過程及臨床意義[J];中國當代兒科雜志;2001年05期

2 趙淑琴;小兒肺炎支原體肺炎有關(guān)的幾個問題[J];小兒急救醫(yī)學雜志;1995年04期

3 馬永臻;睪丸生精細胞凋亡的影響因素[J];中國男科學雜志;2004年01期

4 鄭貞,劉寶紅,刁錫東;支原體肺炎患兒外周血淋巴細胞凋亡的初步觀察[J];山東醫(yī)藥;2000年14期

5 苑同業(yè),刁同進,高百春,劉奉亭,孫英姿,梁冰,禹華瑋;支原體肺炎患者外周血淋巴細胞凋亡過程及臨床意義[J];上海醫(yī)學檢驗雜志;2002年04期

6 董志偉,萬文徽,李振甫,邱萬榮,魏淑敏;抗胃癌細胞系單克隆抗體PD4的初步研究[J];生物化學雜志;1985年02期

7 胡濤,王海燕,高美華,馮獻啟,王運平;沙眼衣原體、溶脲脲原體感染致精漿TNF-α、IL-6升高在男女不育發(fā)病中的意義[J];生殖與避孕;1999年02期

8 楊麥貴;楊陽;郝曉柯;張竹映;鄭善鑾;石繼紅;池巍;;解脲支原體感染的精液中IL-6與生殖細胞凋亡的關(guān)系[J];西北國防醫(yī)學雜志;2006年01期

9 李園;向稚丹;盧建明;;小兒肺炎支原體肺炎免疫功能觀察[J];現(xiàn)代醫(yī)藥衛(wèi)生;2006年13期

10 劉艷紅,李艷,劉先洲,湯紀路;豬鼻支原體抗原體外對白血病細胞增殖及凋亡的影響[J];中華微生物學和免疫學雜志;2003年08期

，

本文編號：1587045