本文關(guān)鍵詞：彎曲菌地方分離株毒力相關(guān)基因分析及HT-29細胞感染模型的建立與應(yīng)用　出處：《揚州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 空腸彎曲菌 毒力相關(guān)基因 HT-29細胞 黏附性 侵襲性 增殖性

【摘要】： 空腸彎曲菌是全球性引起胃腸道感染的主要病原菌之一,并與格林-巴利綜合癥(GBS)及米爾-費歇綜合癥(MFS)等神經(jīng)系統(tǒng)疾病的發(fā)生關(guān)系密切。由于缺乏適當?shù)膶嶒災(zāi)Ｐ?目前對空腸彎曲菌的遺傳學(xué)特性、毒力因子和致病機理了解甚少。本研究的主要內(nèi)容是:(1)對我國部分地區(qū)不同來源的空腸彎曲菌和結(jié)腸彎曲菌分離株毒力相關(guān)基因進行分析;(2)通過建立體外細胞模型,研究空腸彎曲菌分離株的黏附、侵襲和增殖特性,開展空腸彎曲菌的侵襲能力與毒力相關(guān)基因間相互關(guān)系的初步分析。 一、彎曲菌地方分離株毒力相關(guān)基因的分析 為了解我國部分地區(qū)彎曲菌分離株中10種毒力基因的攜帶情況,本研究對分離自腹瀉病人、雞、牛和市場流通的453株空腸彎曲菌和40株結(jié)腸彎曲菌進行基因檢測和分析。結(jié)果表明:結(jié)腸彎曲菌分離株各基因的攜帶率均低于空腸彎曲菌。453株空腸彎曲菌中黏附相關(guān)基因cadF、peb1A、racR平均攜帶率分別為91.39%、95.36%和97.79%,趨化性調(diào)節(jié)基因cheY、docA平均攜帶率分別為95.81%和92.27%,侵襲蛋白基因iamA平均攜帶率為85.87%,鞭毛蛋白基因flaA平均攜帶率為83.66%,而細胞致死性擴張毒素基因cdt、LOS合成相關(guān)基因wlaN、質(zhì)�；騰irB11平均攜帶率僅為72.19%、48.12%和6.57%。同一種基因在不同來源空腸彎曲菌分離株中的分析結(jié)果顯示,人源分離株flaA、virB11基因攜帶率較高,牛源分離株cadF、docA、cheY和iamA基因攜帶率較高,市場分離株peb1A、racR、cdt和wlaN基因攜帶率較高,雞源分離株各基因攜帶率在所有種類分離株中處于中間水平。453株空腸彎曲菌中,86.75%的細菌含有6個以上的毒力相關(guān)基因,但由于不同宿主的特異性,不同來源分離株的毒力基因存在較大差異,由此推測,空腸彎曲菌對不同宿主的致病機理可能存在不同。 二、空腸彎曲菌分離株HT-29細胞感染模型的建立與應(yīng)用 本研究以人結(jié)腸癌細胞系HT-29為材料,建立空腸彎曲菌體外細胞感染模型,用以測定不同來源空腸彎曲菌分離株對HT-29細胞的黏附、侵襲和增殖能力,并對空腸彎曲菌的侵襲能力與毒力基因之間的關(guān)系展開初步分析。42株不同來源的空腸彎曲菌分離株黏附、侵襲和增殖實驗顯示:不同細菌對HT-29細胞的吸附、侵襲和增殖能力存在一定差異,其中11株細菌沒有黏附、侵襲和增殖能力、13株細菌沒有侵襲和增殖能力、3株細菌沒有增殖能力、其余15株細菌同時具有黏附、侵襲和增殖能力,且15株細菌對HT-29細胞的平均黏附效率為0.2270%、平均侵襲效率為0.0778%、平均增殖倍數(shù)為12.55倍。 212株腹瀉病人源和雞源空腸彎曲菌分離株的侵襲力實驗表明:54.72%的細菌無侵襲能力,15.09%的細菌侵襲效率在0.0001%-0.01%之間,14.75%的細菌侵襲效率在0.01%-0.1%之間,12.74%的細菌侵襲效率在0.1%-2%之間。腹瀉病人源分離株侵襲HT-29細胞能力顯著高于雞源分離株。 不同侵襲能力分離株毒力相關(guān)基因分析顯示:cadF、peb1A、docA和flaA基因攜帶率隨著細胞侵襲力的增強而下降,racR、cheY和iamA基因在不同侵襲力菌株間差異不顯著,而virB11、wlaN和cdt等毒素基因攜帶率隨細胞侵襲力的增強而上升。不同侵襲力雞源空腸彎曲菌毒力相關(guān)基因與人源空腸彎曲菌相比,cadF、racR、peb1A、cheY、docA、wlaN和flaA等基因攜帶情況無顯著差異,但iamA、cdt、virB11基因攜帶存在顯著差異,這種差異可能是導(dǎo)致人源分離株HT-29細胞侵襲力高于雞源分離株的原因之一。
[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.

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378

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本文編號：1369901