【摘要】： 目的:A族溶血性鏈球菌(GAS)是咽部、皮膚、軟組織感染的常見致病菌,也是引起兒童中毒性疾病:猩紅熱、毒性休克綜合征等的病原體,它的危害還在于可以導(dǎo)致感染后的自身免疫性疾病、風(fēng)濕熱、風(fēng)濕性心臟病、腎小球腎炎、牛皮癬以及免疫性腦病等。鏈球菌入侵人體后,機(jī)體最初的天然免疫也和抗其他多種病原體一樣,主要是定居的巨噬細(xì)胞和招募來的嗜中性粒細(xì)胞的作用,一些研究已經(jīng)描述了A族鏈球菌通過各種途徑干擾中性粒細(xì)胞的抗微生物活性,包括釋放分子干擾中性粒細(xì)胞的募集,誘導(dǎo)中性粒細(xì)胞死亡等,但是,A族鏈球菌如何逃避巨噬細(xì)胞強(qiáng)大的抗微生物作用國內(nèi)外未有報(bào)道。 本研究旨在探索GAS刺激RAW264.7巨噬細(xì)胞株后,對RAW264.7細(xì)胞的超微結(jié)構(gòu)、細(xì)胞增殖,其表面分子的表達(dá)及其表達(dá)、分泌細(xì)胞因子等的影響,以進(jìn)一步研究GAS的致病及其逃避免疫攻擊的機(jī)制。 方法: 1將GAS分五個(gè)不同時(shí)間段刺激RAW264.7細(xì)胞,同時(shí)細(xì)菌刺激細(xì)胞數(shù)(MOI)分別設(shè)低菌量(2:1、5:1)、中等菌量(10:1、20:1、30:1)和高菌量(50:1、100:1),及PBS陰性和LPS陽性對照,均三復(fù)孔,作用完畢棄菌及加抗生素殺死胞外菌后,于細(xì)胞培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)三天,MTT法檢測刺激前后RAW264.7的增殖情況。 2以低菌量、中等菌量和高菌量GAS刺激細(xì)胞90min時(shí),通過透射電子顯微鏡觀察RAW264.7超微結(jié)構(gòu)的變化,利用TUNEL和ANNEXIN V-FITC細(xì)胞凋亡檢測試劑盒檢測細(xì)胞凋亡情況。 3分別用GAS、大腸桿菌和金黃色葡萄球菌以MOI為5:1時(shí)刺激巨噬細(xì)胞90min,作用完畢繼續(xù)培養(yǎng)三天,行流式細(xì)胞術(shù)檢測RAW264.7細(xì)胞表面CD80、CD86、MHC-Ⅱ的表達(dá)情況;應(yīng)用Real-time-PCR檢測不同刺激組RAW264.7細(xì)胞的IL-1、IL-6、IL-23、TNF-α、TGF-β等炎性細(xì)胞因子的轉(zhuǎn)錄水平變化,并通過ELISA檢測刺激后細(xì)胞分泌IL-6的變化。 4 Real-time-PCR法檢測低菌量GAS刺激小鼠三天后,腹腔巨噬細(xì)胞及脾臟表達(dá)IL-1、IL-6、IL-23、TNF-α、TGF-β的變化。 結(jié)果: 1 MTT結(jié)果顯示,低、中菌量GAS對RAW264.7細(xì)胞增殖有促進(jìn)作用,高菌量GAS對細(xì)胞增殖有抑制作用。 2通過透射電子顯微鏡觀察:低菌量細(xì)菌刺激細(xì)胞時(shí),RAW264.7細(xì)胞的細(xì)胞器發(fā)達(dá),功能旺盛,線粒體水腫,粗面內(nèi)質(zhì)網(wǎng)變粗糙;中劑量刺激時(shí),細(xì)胞器發(fā)達(dá),有髓樣溶酶體結(jié)構(gòu),提示細(xì)胞吞噬消化細(xì)菌不完全;高菌量刺激細(xì)胞時(shí),細(xì)胞出現(xiàn)空泡,核膜不完整等細(xì)胞壞死表象。 3 TUNEL法和ANNEXIN V-FITC法結(jié)果均顯示:高菌量刺激RAW264.7時(shí),細(xì)胞數(shù)減少是由于細(xì)胞壞死而不是凋亡。 4半定量PCR及Real-time-PCR結(jié)果顯示:低菌量GAS刺激RAW264.7時(shí),細(xì)胞表達(dá)IL-1、IL-23及TNF-α基因水平升高,而IL-6、TGF-β水平下降。 5以低菌量GAS、大腸桿菌金和黃色葡萄球菌刺激巨噬細(xì)胞,與對照組相比,各刺激組細(xì)胞表達(dá)CD80分子均升高,且大腸桿菌升高最為顯著,其次分別為金黃色葡萄球菌、GAS;大腸桿菌刺激組CD86表達(dá)水平升高,其余刺激組表達(dá)無明顯變化;GAS刺激組細(xì)胞表面MHC-Ⅱ分子的表達(dá)無顯著變化,但大腸桿菌與金葡菌刺激組表達(dá)升高,且有統(tǒng)計(jì)學(xué)意義。Real-time-PCR結(jié)果顯示:大腸桿菌和金黃色葡萄球菌刺激組RAW264.7表達(dá)IL-6等細(xì)胞因子的轉(zhuǎn)錄均增加,并且大腸桿菌刺激組升高明顯,GAS刺激組IL-6轉(zhuǎn)錄反而下調(diào)。 6通過Real-time-PCR檢測,與對照組比較,低菌量GAS刺激小鼠后,小鼠腹腔巨噬細(xì)胞表達(dá)IL-1、TNF-α水平升高,IL-6、IL-23、TGF-β變化不明顯;小鼠脾細(xì)胞表達(dá)IL-1、IL-6、IL-23、TNF-α、TGF-β水平均增高。 結(jié)論: 1低菌量GAS作用RAW264.7細(xì)胞:能夠促進(jìn)其增殖,使其吞噬功能活躍,其表達(dá)促炎癥因子的基因水平升高,但使其IL-6、TGF-β表達(dá)水平下降,相關(guān)機(jī)制、信號轉(zhuǎn)導(dǎo)通路等仍有待研究。 2低菌量GAS、大腸桿菌、金黃色葡萄球菌能促進(jìn)RAW264.7細(xì)胞表面CD80、MHC-Ⅱ分子表達(dá),說明在一定程度上能增強(qiáng)巨噬細(xì)胞的抗原遞呈作用,但GAS的這種作用不明顯。 3高菌量GAS對細(xì)胞增殖有抑制作用,使巨噬細(xì)胞壞死而不是凋亡。
[Abstract]:Objective: A hemolytic streptococcus (GAS) is a common pathogen of pharynx, skin, and soft tissue infection. It is also a pathogen causing toxic diseases in children: scarlet fever and toxic shock syndrome. Its harm is also caused by autoimmune diseases, rheumatic fever, rheumatic heart disease, glomerulonephritis, psoriasis and immunity after infection. When Streptococcus invades the human body, the original natural immunity of the body is similar to that of many other pathogens, mainly the colonization of macrophages and the recruitment of neutrophils. Some studies have described the anti microbial activity of A Streptococcus through various ways to interfere with neutrophils, including the release of molecular stem cells. The recruitment of neutrophils is disturbed and neutrophil death is induced. However, there is no report on how A Streptococcus can escape the strong anti microbial effects of macrophages at home and abroad.
The purpose of this study was to explore the effects of GAS on the ultrastructure of RAW264.7 cells, cell proliferation, expression and expression of surface molecules, and the secretion of cytokines, so as to further study the pathogenesis of GAS and the mechanism of escaping immune attacks after the stimulation of the RAW264.7 macrophage strain.
Method:
1 RAW264.7 cells were stimulated by GAS in five different time periods, and the number of bacteria stimulating cells (2:1,5:1), medium biomass (10:1,20:1,30:1) and high bacteria quantity (50:1100:1), and PBS negative and LPS positive control were three, both of which were three compound holes. After the effect was finished, the bacteria were abandoned and the extracellular bacteria were killed, and three were continued in the cell culture box. The proliferation of RAW264.7 was detected by MTT before and after stimulation.
2 when the cell 90min was stimulated by low bacteria, medium and high bacteria, the ultrastructure of RAW264.7 was observed by transmission electron microscope, and the apoptosis of the cells was detected by TUNEL and ANNEXIN V-FITC cell apoptosis detection kit.
3 GAS, Escherichia coli and Staphylococcus aureus were used to stimulate macrophage 90min at the time of MOI 5:1. The effect was completed for three days. Flow cytometry was used to detect the expression of CD80, CD86, MHC- II on the surface of RAW264.7 cells, and IL-1, IL-6, IL-6, alpha, beta and other inflammatory cells were detected by Real-time-PCR. The transcription level of the factor was changed, and the changes of IL-6 secretion after stimulation were detected by ELISA.
4 Real-time-PCR method was used to detect the changes of IL-1, IL-6, IL-23, TNF- alpha and TGF- TGF- in peritoneal macrophages and spleen after three days of stimulation with low GAS.
Result:
1 MTT results showed that low and medium bacteria GAS could promote the proliferation of RAW264.7 cells, and high GAS could inhibit cell proliferation.
2 through the transmission electron microscope, the cell organelle of RAW264.7 cells was developed, the function was strong, the mitochondria was edema, the rough endoplasmic reticulum became rough, when the medium dose stimulated, the organelles were developed and the structure of the myeloid lysosome, suggesting that the cells phagocytic bacteria were not complete; when the high amount of bacteria stimulated the cells, the cells appeared vacuoles. Incompleteness of the nuclear membrane and the appearance of cell necrosis.
3 the results of both TUNEL and ANNEXIN V-FITC showed that when RAW264.7 was stimulated by high bacteria, the decrease of cell number was due to cell necrosis rather than apoptosis.
4 semi quantitative PCR and Real-time-PCR results showed that when RAW264.7 was stimulated by low GAS, the expression of IL-1, IL-23 and TNF- alpha gene increased, while IL-6 and TGF- beta levels decreased.
5 the macrophages were stimulated with low bacteria GAS, E. coli gold and Staphylococcus aureus. Compared with the control group, the expression of CD80 molecules in each stimulation group increased and the Escherichia coli was the most significant, followed by Staphylococcus aureus, GAS, and the expression level of CD86 in the Escherichia coli stimulation group, and the expression of the other stimulation groups, and GAS spines. There was no significant change in the expression of MHC- II molecules on the surface of the stimulated group, but the expression of Escherichia coli and Staphylococcus aureus stimulated group increased, and the.Real-time-PCR results showed that the transcription of RAW264.7 expression IL-6 and other cytokines in Escherichia coli and Staphylococcus aureus stimulated group increased, and the Escherichia coli stimulation group increased significantly, GAS stimulation In group IL-6, the transcription was downregulated.
6 compared with the control group, compared with the control group, the mouse peritoneal macrophages expressed IL-1, the level of TNF- a, IL-6, IL-23, TGF- beta were not obvious, and the mice splenocytes expressed IL-1, IL-6, IL-23, TNF- alpha and TGF- beta water increased significantly after the low bacteria amount GAS stimulated the mice.
Conclusion:
1 low bacteria amount GAS acts on RAW264.7 cells: it can promote its proliferation, make its phagocytic function active, and increase the gene level of the expression of pro-inflammatory factors, but the decrease of IL-6, TGF- beta expression level, related mechanism, signal transduction pathway still remain to be studied.
2 low bacteria amount GAS, Escherichia coli and Staphylococcus aureus can promote the expression of CD80 and MHC- II molecules on the surface of RAW264.7 cells, indicating that the antigen presenting effect of macrophages can be enhanced to a certain extent, but the effect of GAS is not obvious.
3 high bacterial GAS inhibited cell proliferation and caused macrophage necrosis rather than apoptosis.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R515;R392

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2 王玉珍;溶酶體相關(guān)的小G蛋白R(shí)ab7b負(fù)向調(diào)節(jié)巨噬細(xì)胞Toll樣受體信號轉(zhuǎn)導(dǎo)的研究[D];浙江大學(xué);2006年

3 楊洋;DcR3干預(yù)肺纖維化動(dòng)物模型及其轉(zhuǎn)染人成纖維細(xì)胞的實(shí)驗(yàn)研究[D];吉林大學(xué);2009年

4 馬翠卿;GAS下調(diào)巨噬細(xì)胞炎癥因子的作用及機(jī)制研究[D];河北醫(yī)科大學(xué);2011年

5 鞠瑞;羧胺三唑的抗癌新機(jī)制抑制腫瘤相關(guān)巨噬細(xì)胞中促炎細(xì)胞因子的釋放[D];北京協(xié)和醫(yī)學(xué)院;2011年

6 尚Z腪，

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