弯曲菌地方分离株毒力相关基因分析及HT-29细胞感染模型的建立与应用

发布时间：2018-01-02 15:46

本文关键词：弯曲菌地方分离株毒力相关基因分析及HT-29细胞感染模型的建立与应用　出处：《扬州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 空肠弯曲菌是全球性引起胃肠道感染的主要病原菌之一,并与格林-巴利综合症(GBS)及米尔-费歇综合症(MFS)等神经系统疾病的发生关系密切。由于缺乏适当的实验模型,目前对空肠弯曲菌的遗传学特性、毒力因子和致病机理了解甚少。本研究的主要内容是:(1)对我国部分地区不同来源的空肠弯曲菌和结肠弯曲菌分离株毒力相关基因进行分析;(2)通过建立体外细胞模型,研究空肠弯曲菌分离株的黏附、侵袭和增殖特性,开展空肠弯曲菌的侵袭能力与毒力相关基因间相互关系的初步分析。一、弯曲菌地方分离株毒力相关基因的分析为了解我国部分地区弯曲菌分离株中10种毒力基因的携带情况,本研究对分离自腹泻病人、鸡、牛和市场流通的453株空肠弯曲菌和40株结肠弯曲菌进行基因检测和分析。结果表明:结肠弯曲菌分离株各基因的携带率均低于空肠弯曲菌。453株空肠弯曲菌中黏附相关基因cadF、peb1A、racR平均携带率分别为91.39%、95.36%和97.79%,趋化性调节基因cheY、docA平均携带率分别为95.81%和92.27%,侵袭蛋白基因iamA平均携带率为85.87%,鞭毛蛋白基因flaA平均携带率为83.66%,而细胞致死性扩张毒素基因cdt、LOS合成相关基因wlaN、质粒基因virB11平均携带率仅为72.19%、48.12%和6.57%。同一种基因在不同来源空肠弯曲菌分离株中的分析结果显示,人源分离株flaA、virB11基因携带率较高,牛源分离株cadF、docA、cheY和iamA基因携带率较高,市场分离株peb1A、racR、cdt和wlaN基因携带率较高,鸡源分离株各基因携带率在所有种类分离株中处于中间水平。453株空肠弯曲菌中,86.75%的细菌含有6个以上的毒力相关基因,但由于不同宿主的特异性,不同来源分离株的毒力基因存在较大差异,由此推测,空肠弯曲菌对不同宿主的致病机理可能存在不同。二、空肠弯曲菌分离株HT-29细胞感染模型的建立与应用本研究以人结肠癌细胞系HT-29为材料,建立空肠弯曲菌体外细胞感染模型,用以测定不同来源空肠弯曲菌分离株对HT-29细胞的黏附、侵袭和增殖能力,并对空肠弯曲菌的侵袭能力与毒力基因之间的关系展开初步分析。42株不同来源的空肠弯曲菌分离株黏附、侵袭和增殖实验显示:不同细菌对HT-29细胞的吸附、侵袭和增殖能力存在一定差异,其中11株细菌没有黏附、侵袭和增殖能力、13株细菌没有侵袭和增殖能力、3株细菌没有增殖能力、其余15株细菌同时具有黏附、侵袭和增殖能力,且15株细菌对HT-29细胞的平均黏附效率为0.2270%、平均侵袭效率为0.0778%、平均增殖倍数为12.55倍。 212株腹泻病人源和鸡源空肠弯曲菌分离株的侵袭力实验表明:54.72%的细菌无侵袭能力,15.09%的细菌侵袭效率在0.0001%-0.01%之间,14.75%的细菌侵袭效率在0.01%-0.1%之间,12.74%的细菌侵袭效率在0.1%-2%之间。腹泻病人源分离株侵袭HT-29细胞能力显著高于鸡源分离株。不同侵袭能力分离株毒力相关基因分析显示:cadF、peb1A、docA和flaA基因携带率随着细胞侵袭力的增强而下降,racR、cheY和iamA基因在不同侵袭力菌株间差异不显著,而virB11、wlaN和cdt等毒素基因携带率随细胞侵袭力的增强而上升。不同侵袭力鸡源空肠弯曲菌毒力相关基因与人源空肠弯曲菌相比,cadF、racR、peb1A、cheY、docA、wlaN和flaA等基因携带情况无显著差异,但iamA、cdt、virB11基因携带存在显著差异,这种差异可能是导致人源分离株HT-29细胞侵袭力高于鸡源分离株的原因之一。
[Abstract]:Campylobacter jejuni is a global one of the main pathogens causing gastrointestinal infection bacteria, and Green Barre syndrome (GBS) and Mir Fisher syndrome (MFS) and other neurological diseases are closely related. Due to the lack of appropriate experimental model, the Campylobacter jejuni genetics characteristics, virulence factors and understanding the mechanism is very few. The main contents of this study are: (1) analyze in some regions of different sources of Campylobacter spp. isolates of Virulence Related Genes; (2) through the establishment of a cell model in vitro, Campylobacter jejuni isolates of adhesion, invasion and proliferation characteristics, a preliminary analysis of the relationship between the invasion ability and Virulence Related Genes of Campylobacter jejuni.
Analysis of Virulence Related Genes in local isolates of Campylobacter
In order to understand the carrying situation in some areas of China Campylobacter isolates 10 virulence genes, this study isolated from patients with diarrhea, chicken, cattle and gene detection and analysis of the market circulation of 453 strains of Campylobacter jejuni and 40 strains of Campylobacter coli. The results showed that: Campylobacter isolates the gene carrying rate the adhesion was lower than.453 of Campylobacter jejuni strains of Campylobacter jejuni in related genes cadF, peb1A, racR average carrying rate were 91.39%, 95.36% and 97.79%, chemotaxis regulation gene cheY, docA average carrying rate were 95.81% and 92.27%, invasive protein gene iamA carried an average rate of 85.87%, the average flaA flagellin gene carrying rate 83.66%, while the cytolethal distending toxin gene CDT, LOS synthesis related gene wlaN, virB11 gene plasmid carried an average rate of only 72.19%, 48.12% and 6.57%. with a gene in different sources of Campylobacter jejuni isolates The analysis results showed that the strains isolated from human flaA, higher positive rate of virB11 gene, bovine strains cadF, docA, cheY and iamA gene carrying rate is higher, the market strains peb1A, racR, CDT and higher positive rate of wlaN gene from chicken isolates of each gene carrying rate of isolates in the middle level of.453 strains of Campylobacter jejuni in all 86.75% species, the bacteria contain Virulence Related Genes of more than 6, but due to different host specificity, there are differences between different sources of virulence genes of isolates thus speculated that Campylobacter jejuni on different host pathogenic mechanism may be different.
Two, establishment and application of HT-29 cell infection model of Campylobacter jejuni isolate
In this study, human colon cancer cell line HT-29, the establishment of Campylobacter jejuni in vitro infection model was used to test the different sources of Campylobacter jejuni isolates on HT-29 cell adhesion, proliferation and invasion, and the relationship between invasiveness and virulence gene of Campylobacter jejuni to the preliminary analysis of.42 strains of Campylobacter jejuni different sources of isolates showed adhesion, invasion and proliferation of experimental adsorption of different bacteria on HT-29 cells, there are some differences in proliferation and invasion, including 11 strains of bacteria without adhesion, proliferation and invasion, 13 strains of bacteria have proliferation and invasion, 3 strains of bacteria have the ability of proliferation with the remaining 15 strains of bacteria the adhesion, proliferation and invasion, and 15 strains of bacterial adhesion to HT-29 cells the average efficiency was 0.2270%, the average invasion rate was 0.0778%, the average proliferation multiples of 12.55.
Showed that 212 strains of diarrhea patients and source of avian Campylobacter isolates invasion experiment: 54.72% no bacteria invasion, 15.09% bacterial invasion efficiency between 0.0001%-0.01%, 14.75% of the bacterial invasion efficiency between 0.01%-0.1%, 12.74% of the bacterial invasion efficiency between 0.1%-2% strains isolated from diarrhea patients. Invasion ability of HT-29 cells significantly higher than the chicken isolates.
The invasion ability of different strains separation and analysis of virulence related genes: cadF, peb1A, docA and flaA gene carrying rate with the increase of cell invasiveness decreased, racR, cheY and iamA genes in different invasion strains had no significant difference, while virB11, wlaN and CDT toxin genes with enhanced rate of cell invasion and rise. Compared with different invasiveness of avian Campylobacter jejuni virulence related genes and human Campylobacter jejuni cadF, racR, peb1A, cheY, docA, wlaN and flaA genes had no significant difference, but iamA, CDT, virB11 gene has significant difference to carry, this difference may be caused human isolate HT-29 one of the reasons for cell invasion than chicken isolates.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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