GAS对巨噬细胞株RAW264.7功能的影响

发布时间：2018-07-28 08:57

【摘要】： 目的:A族溶血性链球菌(GAS)是咽部、皮肤、软组织感染的常见致病菌,也是引起儿童中毒性疾病:猩红热、毒性休克综合征等的病原体,它的危害还在于可以导致感染后的自身免疫性疾病、风湿热、风湿性心脏病、肾小球肾炎、牛皮癣以及免疫性脑病等。链球菌入侵人体后,机体最初的天然免疫也和抗其他多种病原体一样,主要是定居的巨噬细胞和招募来的嗜中性粒细胞的作用,一些研究已经描述了A族链球菌通过各种途径干扰中性粒细胞的抗微生物活性,包括释放分子干扰中性粒细胞的募集,诱导中性粒细胞死亡等,但是,A族链球菌如何逃避巨噬细胞强大的抗微生物作用国内外未有报道。本研究旨在探索GAS刺激RAW264.7巨噬细胞株后,对RAW264.7细胞的超微结构、细胞增殖,其表面分子的表达及其表达、分泌细胞因子等的影响,以进一步研究GAS的致病及其逃避免疫攻击的机制。方法: 1将GAS分五个不同时间段刺激RAW264.7细胞,同时细菌刺激细胞数(MOI)分别设低菌量(2:1、5:1)、中等菌量(10:1、20:1、30:1)和高菌量(50:1、100:1),及PBS阴性和LPS阳性对照,均三复孔,作用完毕弃菌及加抗生素杀死胞外菌后,于细胞培养箱内继续培养三天,MTT法检测刺激前后RAW264.7的增殖情况。 2以低菌量、中等菌量和高菌量GAS刺激细胞90min时,通过透射电子显微镜观察RAW264.7超微结构的变化,利用TUNEL和ANNEXIN V-FITC细胞凋亡检测试剂盒检测细胞凋亡情况。 3分别用GAS、大肠杆菌和金黄色葡萄球菌以MOI为5:1时刺激巨噬细胞90min,作用完毕继续培养三天,行流式细胞术检测RAW264.7细胞表面CD80、CD86、MHC-Ⅱ的表达情况;应用Real-time-PCR检测不同刺激组RAW264.7细胞的IL-1、IL-6、IL-23、TNF-α、TGF-β等炎性细胞因子的转录水平变化,并通过ELISA检测刺激后细胞分泌IL-6的变化。 4 Real-time-PCR法检测低菌量GAS刺激小鼠三天后,腹腔巨噬细胞及脾脏表达IL-1、IL-6、IL-23、TNF-α、TGF-β的变化。结果: 1 MTT结果显示,低、中菌量GAS对RAW264.7细胞增殖有促进作用,高菌量GAS对细胞增殖有抑制作用。 2通过透射电子显微镜观察:低菌量细菌刺激细胞时,RAW264.7细胞的细胞器发达,功能旺盛,线粒体水肿,粗面内质网变粗糙;中剂量刺激时,细胞器发达,有髓样溶酶体结构,提示细胞吞噬消化细菌不完全;高菌量刺激细胞时,细胞出现空泡,核膜不完整等细胞坏死表象。 3 TUNEL法和ANNEXIN V-FITC法结果均显示:高菌量刺激RAW264.7时,细胞数减少是由于细胞坏死而不是凋亡。 4半定量PCR及Real-time-PCR结果显示:低菌量GAS刺激RAW264.7时,细胞表达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、大肠杆菌金和黄色葡萄球菌刺激巨噬细胞,与对照组相比,各刺激组细胞表达CD80分子均升高,且大肠杆菌升高最为显著,其次分别为金黄色葡萄球菌、GAS;大肠杆菌刺激组CD86表达水平升高,其余刺激组表达无明显变化;GAS刺激组细胞表面MHC-Ⅱ分子的表达无显著变化,但大肠杆菌与金葡菌刺激组表达升高,且有统计学意义。Real-time-PCR结果显示:大肠杆菌和金黄色葡萄球菌刺激组RAW264.7表达IL-6等细胞因子的转录均增加,并且大肠杆菌刺激组升高明显,GAS刺激组IL-6转录反而下调。 6通过Real-time-PCR检测,与对照组比较,低菌量GAS刺激小鼠后,小鼠腹腔巨噬细胞表达IL-1、TNF-α水平升高,IL-6、IL-23、TGF-β变化不明显;小鼠脾细胞表达IL-1、IL-6、IL-23、TNF-α、TGF-β水平均增高。结论: 1低菌量GAS作用RAW264.7细胞:能够促进其增殖,使其吞噬功能活跃,其表达促炎症因子的基因水平升高,但使其IL-6、TGF-β表达水平下降,相关机制、信号转导通路等仍有待研究。 2低菌量GAS、大肠杆菌、金黄色葡萄球菌能促进RAW264.7细胞表面CD80、MHC-Ⅱ分子表达,说明在一定程度上能增强巨噬细胞的抗原递呈作用,但GAS的这种作用不明显。 3高菌量GAS对细胞增殖有抑制作用,使巨噬细胞坏死而不是凋亡。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R515;R392

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