致肾盂肾炎大肠杆菌与细胞相互作用的初步研究
发布时间：2018-07-29 07:26
【摘要】： 目的： 建立致肾盂肾炎大肠杆菌(pyelonephritic E．coli or uropathogenic E．coli，UPEC)粘附细胞模型，了解细胞表面粘附受体分布情况，并检测膀胱癌EJ细胞感染UPEC132后基因表达谱变化。 方法： 1．细胞培养：非洲绿猴肾细胞(Vero细胞)、人肾癌细胞系(Ketr-3细胞)、膀胱癌细胞系(EJ细胞)。比较UPEC132对此3种细胞的粘附能力。经Giemsa染色观察UPEC132粘附后不同细胞的形态学变化，计数并统计不同细胞的粘附率及粘附指数。并进行UPEC132的侵袭试验，用庆大霉素杀死细胞外菌后计数胞内菌，统计UPEC132对不同细胞的侵袭指数。同时比较无菌毛代表株E．coli K-12 p678-54的粘附及侵袭能力。 2．运用间接免疫荧光法检测上述3种细胞表面的P菌毛受体，一抗选用抗P_1抗体(小鼠单克隆抗体IgM)，二抗为FITC(异硫氰酸荧光素)标记的兔抗小鼠IgM抗体，在荧光显微镜下观察细胞表面荧光特点，比较其受体分布情况。 3．基因芯片检测膀胱癌EJ细胞感染UPEC132后基因表达谱变化。用Trizol提取EJ细胞总RNA(感染后细胞设为实验组，未感染细胞设为对照组)，并对总RNA进行浓缩、定量、质检，然后利用cRNA标记方法对样品进行荧光标记，纯化后用于芯片杂交；用LuxScan 10K／A双通道激光扫描仪(CapitalBio公司)进行芯片扫描，采用LuxScan3．0图像分析软件(CapitalBio公司)对芯片图像进行分析，把图像信号转化为数字信号；然后对芯片上的数据用Lowess方法进行归一化；最后以差异为2倍的标准来确定差异表达基因。 结果： 1．UPEC132作用于上述3种细胞1h后，细胞形态均发生了明显改变：细胞间隙变大，胞浆突起收缩，甚至破裂，细胞对培养皿的粘附能力也明显下降。UPEC132对Vero、Ketr-3及EJ细胞的粘附率分别为(61．44±3．21)％、(55．22±4．09)％及(58．67±5．12)％，差别无统计学意义；对此3种细胞的粘附指数分别为1．44±0．06、1．74±0．09和2．27±0．18，有统计学差异(p0．05)。UPEC132对EJ及Ketr-3细胞的侵袭指数分别为(3．25±0．20)×10~(-3)、(3．00±0．34)×10~(-3)，两者之间无统计学差异，但均高于对Vero细胞的侵袭指数[(2．61±0．32)×10~(-3)，p0．05]。E．coli K-12 p678-54对上述3种细胞无粘附及侵袭能力，细胞形态未见改变。 2．荧光显微镜下实验组细胞表面呈黄绿色荧光，细胞轮廓清晰，荧光物质在细胞表面呈连续性分布。而对照组未见细胞表面荧光。提示此3种细胞表面存在P菌毛受体。 3．膀胱癌EJ细胞感染UPEC132后的基因表达谱检测共发现29个差异表达基因，其中28个基因表达上调，1个基因表达下调，这些差异表达基因主要涉及细胞生长与增殖、炎症反应、细胞凋亡、应激反应、信号转导等方面，表明UPEC132粘附EJ细胞后在多靶点、多层次、多通路与宿主细胞发生相互作用。 结论： 本研究建立了UPEC132感染细胞的模型，分析了该菌的粘附及侵袭能力，检测了被感染细胞表面受体分布情况，并用基因芯片技术检测EJ细胞感染UPEC132后基因表达谱变化，为致肾盂肾炎大肠杆菌的致病机理及其与细胞相互作用分子机制的深入研究奠定了良好的基础。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.21
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