致腎盂腎炎大腸桿菌與細胞相互作用的初步研究
發(fā)布時間：2018-07-29 07:26
【摘要】： 目的： 建立致腎盂腎炎大腸桿菌(pyelonephritic E．coli or uropathogenic E．coli，UPEC)粘附細胞模型，了解細胞表面粘附受體分布情況，并檢測膀胱癌EJ細胞感染UPEC132后基因表達譜變化。 方法： 1．細胞培養(yǎng)：非洲綠猴腎細胞(Vero細胞)、人腎癌細胞系(Ketr-3細胞)、膀胱癌細胞系(EJ細胞)。比較UPEC132對此3種細胞的粘附能力。經(jīng)Giemsa染色觀察UPEC132粘附后不同細胞的形態(tài)學(xué)變化，計數(shù)并統(tǒng)計不同細胞的粘附率及粘附指數(shù)。并進行UPEC132的侵襲試驗，用慶大霉素殺死細胞外菌后計數(shù)胞內(nèi)菌，統(tǒng)計UPEC132對不同細胞的侵襲指數(shù)。同時比較無菌毛代表株E．coli K-12 p678-54的粘附及侵襲能力。 2．運用間接免疫熒光法檢測上述3種細胞表面的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胞表面熒光特點，比較其受體分布情況。 3．基因芯片檢測膀胱癌EJ細胞感染UPEC132后基因表達譜變化。用Trizol提取EJ細胞總RNA(感染后細胞設(shè)為實驗組，未感染細胞設(shè)為對照組)，并對總RNA進行濃縮、定量、質(zhì)檢，然后利用cRNA標(biāo)記方法對樣品進行熒光標(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)進行芯片掃描，采用LuxScan3．0圖像分析軟件(CapitalBio公司)對芯片圖像進行分析，把圖像信號轉(zhuǎn)化為數(shù)字信號；然后對芯片上的數(shù)據(jù)用Lowess方法進行歸一化；最后以差異為2倍的標(biāo)準(zhǔn)來確定差異表達基因。 結(jié)果： 1．UPEC132作用于上述3種細胞1h后，細胞形態(tài)均發(fā)生了明顯改變：細胞間隙變大，胞漿突起收縮，甚至破裂，細胞對培養(yǎng)皿的粘附能力也明顯下降。UPEC132對Vero、Ketr-3及EJ細胞的粘附率分別為(61．44±3．21)％、(55．22±4．09)％及(58．67±5．12)％，差別無統(tǒng)計學(xué)意義；對此3種細胞的粘附指數(shù)分別為1．44±0．06、1．74±0．09和2．27±0．18，有統(tǒng)計學(xué)差異(p0．05)。UPEC132對EJ及Ketr-3細胞的侵襲指數(shù)分別為(3．25±0．20)×10~(-3)、(3．00±0．34)×10~(-3)，兩者之間無統(tǒng)計學(xué)差異，但均高于對Vero細胞的侵襲指數(shù)[(2．61±0．32)×10~(-3)，p0．05]。E．coli K-12 p678-54對上述3種細胞無粘附及侵襲能力，細胞形態(tài)未見改變。 2．熒光顯微鏡下實驗組細胞表面呈黃綠色熒光，細胞輪廓清晰，熒光物質(zhì)在細胞表面呈連續(xù)性分布。而對照組未見細胞表面熒光。提示此3種細胞表面存在P菌毛受體。 3．膀胱癌EJ細胞感染UPEC132后的基因表達譜檢測共發(fā)現(xiàn)29個差異表達基因，其中28個基因表達上調(diào)，1個基因表達下調(diào)，這些差異表達基因主要涉及細胞生長與增殖、炎癥反應(yīng)、細胞凋亡、應(yīng)激反應(yīng)、信號轉(zhuǎn)導(dǎo)等方面，表明UPEC132粘附EJ細胞后在多靶點、多層次、多通路與宿主細胞發(fā)生相互作用。 結(jié)論： 本研究建立了UPEC132感染細胞的模型，分析了該菌的粘附及侵襲能力，檢測了被感染細胞表面受體分布情況，并用基因芯片技術(shù)檢測EJ細胞感染UPEC132后基因表達譜變化，為致腎盂腎炎大腸桿菌的致病機理及其與細胞相互作用分子機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)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378.21
[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)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378.21
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