【摘要】： 目的： 建立致腎盂腎炎大腸桿菌(pyelonephritic E．coli or uropathogenic E．coli，UPEC)粘附細(xì)胞模型，了解細(xì)胞表面粘附受體分布情況，并檢測(cè)膀胱癌EJ細(xì)胞感染UPEC132后基因表達(dá)譜變化。 方法： 1．細(xì)胞培養(yǎng)：非洲綠猴腎細(xì)胞(Vero細(xì)胞)、人腎癌細(xì)胞系(Ketr-3細(xì)胞)、膀胱癌細(xì)胞系(EJ細(xì)胞)。比較UPEC132對(duì)此3種細(xì)胞的粘附能力。經(jīng)Giemsa染色觀察UPEC132粘附后不同細(xì)胞的形態(tài)學(xué)變化，計(jì)數(shù)并統(tǒng)計(jì)不同細(xì)胞的粘附率及粘附指數(shù)。并進(jìn)行UPEC132的侵襲試驗(yàn)，用慶大霉素殺死細(xì)胞外菌后計(jì)數(shù)胞內(nèi)菌，統(tǒng)計(jì)UPEC132對(duì)不同細(xì)胞的侵襲指數(shù)。同時(shí)比較無(wú)菌毛代表株E．coli K-12 p678-54的粘附及侵襲能力。 2．運(yùn)用間接免疫熒光法檢測(cè)上述3種細(xì)胞表面的P菌毛受體，一抗選用抗P_1抗體(小鼠單克隆抗體IgM)，二抗為FITC(異硫氰酸熒光素)標(biāo)記的兔抗小鼠IgM抗體，在熒光顯微鏡下觀察細(xì)胞表面熒光特點(diǎn)，比較其受體分布情況。 3．基因芯片檢測(cè)膀胱癌EJ細(xì)胞感染UPEC132后基因表達(dá)譜變化。用Trizol提取EJ細(xì)胞總RNA(感染后細(xì)胞設(shè)為實(shí)驗(yàn)組，未感染細(xì)胞設(shè)為對(duì)照組)，并對(duì)總RNA進(jìn)行濃縮、定量、質(zhì)檢，然后利用cRNA標(biāo)記方法對(duì)樣品進(jìn)行熒光標(biāo)記，純化后用于芯片雜交；用LuxScan 10K／A雙通道激光掃描儀(CapitalBio公司)進(jìn)行芯片掃描，采用LuxScan3．0圖像分析軟件(CapitalBio公司)對(duì)芯片圖像進(jìn)行分析，把圖像信號(hào)轉(zhuǎn)化為數(shù)字信號(hào)；然后對(duì)芯片上的數(shù)據(jù)用Lowess方法進(jìn)行歸一化；最后以差異為2倍的標(biāo)準(zhǔn)來(lái)確定差異表達(dá)基因。 結(jié)果： 1．UPEC132作用于上述3種細(xì)胞1h后，細(xì)胞形態(tài)均發(fā)生了明顯改變：細(xì)胞間隙變大，胞漿突起收縮，甚至破裂，細(xì)胞對(duì)培養(yǎng)皿的粘附能力也明顯下降。UPEC132對(duì)Vero、Ketr-3及EJ細(xì)胞的粘附率分別為(61．44±3．21)％、(55．22±4．09)％及(58．67±5．12)％，差別無(wú)統(tǒng)計(jì)學(xué)意義；對(duì)此3種細(xì)胞的粘附指數(shù)分別為1．44±0．06、1．74±0．09和2．27±0．18，有統(tǒng)計(jì)學(xué)差異(p0．05)。UPEC132對(duì)EJ及Ketr-3細(xì)胞的侵襲指數(shù)分別為(3．25±0．20)×10~(-3)、(3．00±0．34)×10~(-3)，兩者之間無(wú)統(tǒng)計(jì)學(xué)差異，但均高于對(duì)Vero細(xì)胞的侵襲指數(shù)[(2．61±0．32)×10~(-3)，p0．05]。E．coli K-12 p678-54對(duì)上述3種細(xì)胞無(wú)粘附及侵襲能力，細(xì)胞形態(tài)未見(jiàn)改變。 2．熒光顯微鏡下實(shí)驗(yàn)組細(xì)胞表面呈黃綠色熒光，細(xì)胞輪廓清晰，熒光物質(zhì)在細(xì)胞表面呈連續(xù)性分布。而對(duì)照組未見(jiàn)細(xì)胞表面熒光。提示此3種細(xì)胞表面存在P菌毛受體。 3．膀胱癌EJ細(xì)胞感染UPEC132后的基因表達(dá)譜檢測(cè)共發(fā)現(xiàn)29個(gè)差異表達(dá)基因，其中28個(gè)基因表達(dá)上調(diào)，1個(gè)基因表達(dá)下調(diào)，這些差異表達(dá)基因主要涉及細(xì)胞生長(zhǎng)與增殖、炎癥反應(yīng)、細(xì)胞凋亡、應(yīng)激反應(yīng)、信號(hào)轉(zhuǎn)導(dǎo)等方面，表明UPEC132粘附EJ細(xì)胞后在多靶點(diǎn)、多層次、多通路與宿主細(xì)胞發(fā)生相互作用。 結(jié)論： 本研究建立了UPEC132感染細(xì)胞的模型，分析了該菌的粘附及侵襲能力，檢測(cè)了被感染細(xì)胞表面受體分布情況，并用基因芯片技術(shù)檢測(cè)EJ細(xì)胞感染UPEC132后基因表達(dá)譜變化，為致腎盂腎炎大腸桿菌的致病機(jī)理及其與細(xì)胞相互作用分子機(jī)制的深入研究奠定了良好的基礎(chǔ)。
[Abstract]:Objective:
To establish the adhesion cell model of pyelonephritic E.coli or uropathogenic E.coli (UPEC), to understand the distribution of adhesion receptors on the cell surface, and to detect the gene expression profiles of EJ cells infected with UPEC132 in bladder cancer after UPEC132.
Method:
1. cell culture: African green monkey kidney cell (Vero cell), human kidney cancer cell line (Ketr-3 cell) and bladder cancer cell line (EJ cell). Compare the adhesion ability of 3 kinds of cells. After Giemsa staining, the morphological changes of different cells after UPEC132 adhesion were observed, and the adhesion rate and adhesion index of different cells were counted, and UPEC132 was counted and UPEC132 was counted. The invasion test was carried out with gentamicin killing extracellular bacteria and counting the invasion index of UPEC132 to different cells. Meanwhile, the adhesion and invasion ability of E.coli K-12 p678-54 of aseptic hair plant was compared.
2. the P pili receptor on the surface of the 3 cells was detected by indirect immunofluorescence, one anti P_1 antibody (murine monoclonal antibody IgM) and two anti IgM antibody labeled with FITC (fluorescein isothiocyanate) in rabbits. The fluorescence special point of the cell surface was observed under the fluorescence microscope, and the distribution of its receptor was compared.
3. gene chip was used to detect the change of gene expression profiles after UPEC132 infection of EJ cells in bladder cancer cells. The total RNA of EJ cells was extracted with Trizol (the infected cells were set as the experimental group, the uninfected cells were set as the control group), and the total RNA was concentrated, quantified, and tested, and then the samples were labeled with cRNA labeling method and used for chip hybridization; L was used for L. L UxScan 10K / A dual channel laser scanner (CapitalBio) carries out chip scanning, analyzes chip images by LuxScan3.0 image analysis software (CapitalBio company), transforms image signals into digital signals, and then uses Lowess method to normalize the data on the chip; finally, the difference is 2 times the standard to determine the difference. Different expression genes.
Result:
After the action of 1.UPEC132 on the 3 kinds of cells, 1H, the cell morphology changed obviously: the cell gap became larger, the cytoplasmic protuberance contracted and even ruptured. The adhesion of cell to the culture dish also decreased significantly by.UPEC132 to Vero, Ketr-3 and EJ cells (55.22 + 3.21)%, (55.22 + 4.09)% and (58.67 + 5.12)%, respectively. There was no statistical difference, the adhesion index of the 3 cells were 1.44 + 0.06,1.74 + 0.09 and 2.27 + 0.18 respectively, and the invasion index of EJ and Ketr-3 cells was (3.25 + 0.20) x 10~ (-3) and (3 + 0.34) x 10~ (-3), respectively. There was no statistical difference between them, but they were all higher than those of Vero. The invasion index of cells was (2.61 + 0.32) x 10~ (-3), and p0.05].E.coli K-12 p678-54 had no adhesion and invasion ability to the 3 kinds of cells, and the cell morphology did not change.
In the 2. fluorescence microscope, the cell surface of the experimental group was yellow green fluorescence, the cell outline was clear, the fluorescent substance was continuously distributed on the cell surface, while the control group did not have the cell surface fluorescence. It suggested that the surface of the 3 cells existed the P pilus receptor.
3. the gene expression profiles of 3. bladder cancer cells infected with UPEC132 were detected in 29 differentially expressed genes, of which 28 genes were up-regulated and 1 genes were down regulated. These differentially expressed genes were mainly involved in cell growth and proliferation, inflammatory reaction, cell apoptosis, stress response, signal transduction and so on, indicating that UPEC132 adhered to EJ cells. Multi target, multilevel and multi-channel interaction with host cells.
Conclusion:
In this study, we established a model of UPEC132 infected cells, analyzed the adhesion and invasion ability of the bacteria, detected the distribution of the receptor on the surface of the infected cells, and detected the gene expression profiles of the EJ cells infected with UPEC132 by gene chip technology, in order to cause the pathogenesis of pyelonephritis and the molecular mechanism of the cell interaction. The deep research laid a good foundation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378.21

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