小探对人膀胱移行上皮细胞基因表达谱

1、小探对人膀胱移行上皮细胞基因表达谱    Effects of uropathogenic Escherichia coli infection on gene expression profiles in human bladder transitional epithelial cells Abstract Interaction between uropathogenic Escherichia coli (UPEC) and host uroepithelial cells is the key step of theprocess of uri

2、nary tract infection. To further define the role uroepithelial cells play in initiating and modulatingthe host response to infection with UPEC strains, the human bladder transitional epithelial EJ cells wereevaluated for their capacities to allow the adherence and invasion by UPEC132, a clinical str

3、ain isolated fromChina, and a cDNA microarray for 22 000 human genes was used to identify the gene expression differencesbetween EJ cells infected with UPEC132 and uninfected EJ cells. Microscope observation showed thatUPEC132 could adhere to EJ cells, and visualization by confocal microscope reveal

4、ed that this invasivemicroorganism could be seen within the cells. EJ cells infected with UPEC132 significantly changed mRNAexpression of a total of 29 genes, including 28 genes up-regulated and 1 gene down-regulated. Of these,regulators of growth and proliferation (e.g. immediate-early genes), cyto

5、kines (e.g. interleukin-6, interleukin-8),and modulators of apoptotic responses were the most prominent. In addition, the deduced signal transductionevents based on bioinformatics analysis disclosed the complicated interaction between UPEC and host cells. Key words: uropathogenic Escherichia coli; g

6、ene expression; bladder epithelial cell; cDNA microarray    1 Introduction Urinary tract infection (UTI) is one of the most common infectious diseases. Each year, an estimated 150 millionhumans worldwide develop UTI. The majority of UTIs are caused by uropathogenic strains of Esc

7、herichia coli(UPEC). UPEC are distinct from most diarrheagenic or commensal Escherichia coli strains because theyproduce a number of UTI-associated virulence factors that permit their successful colonization in the urinarytract. A range of putative and established virulence genes have been identifie

8、d in UPEC that enable these strainsto overcome host defenses and establish infection in this unique niche. These factors include fimbrial adhesins(e.g. type 1 and P pili), toxins (e.g. cytotoxic necrotizing factor 1, hemolysin, and secreted autotransporter toxin),host defense avoidance mechanisms (e

9、.g. capsule), and multiple iron acquisition systems (e.g. aerobactin,enterobactin and yersiniabactin) 3,4. Especially for P and type 1 pili, the attachment to uroepithelial cellsmediated by the two adhesins is the decisive factor of causing the ascending infection. The adherence mediated by pili is

10、the key initial step of activating host cell responses. Through animal modelsof urinary tract infection, researchers have found that P pili-mediated attachment induces epithelial cellactivation such as recruitment of the toll-like receptor 4, production of cytokines like IL-6 and the IL-8, andneutro

11、phil chemotaxis. Type 1 pili can also directly stimulate host cell signaling cascades that lead to theinduction of cytoskeletal rearrangements and the internalization of adherent UPEC, which allows pathogensto evade host defenses. Therefore, it is an important method for exploring bacterial pathogen

12、ic mechanism tostudy the interaction between UPEC and host cells. The UPEC-uroepithelium interaction must lead to activation of a program of epithelial gene expression. However, only a few differentially expressed genes such as several cell receptors, cytokines and signal moleculeshave been characte

13、rized in uroepithelial cells infected with UPEC, which probably represent only a smallfraction of all the genes that are induced under these circumstances. Consequently, identification of all thesedifferential expressed genes, and ultimately characterization of their functions, will be important for

14、understanding the pathogenesis of infections with uropathogens. In this report, we describe how a functionalgenomics-based analysis of the cell response to a clinical UPEC132 strain infection of the human bladderepithelial cell EJ has revealed an intricate network of molecular regulators and effecto

15、rs of uroepithelial growthand proliferation, pro-inflammatory responses, and apoptosis.    2 Materials and methods 2.1 Bacterial strains and plasmids UPEC132 was isolated from the urine of a patient with acute pyelonephritis. The major P pili structuralsubunit PapA of this strain

16、 was serotype F13, and the top adhesin PapG was class II, which were quite differentfrom UPEC model strains J96 (F13 PapA plus PapG I/III), 536 (F536 PapA plus PapG III) and CFT073(F7-1/F7-2 PapA plus PapG II). All strains were routinely grown at 37°C in Luria-Bertani (LB) broth or onLB agar. P

17、lasmid pSELECT-GFPzeo-mcs (Invivogen, CAUSA) contained the gene encoding green fluorescentprotein (GFP). 2.2 Cell culture The human epithelial EJ cell (derived from bladder carcinoma), provided by Tianjin Cancer Institute, weregrown in Dulbecco's modified eagle medium(Sigma, MO, USA) supplemente

18、d with 10% fetal bovine serum at37°C with 5% CO2. 2.3 Microscope observation for bacterial adherence EJ cells were grown to confluence in 90mm culture dishes (Corning, NY, USA). Just before infection, the cellculture medium in each dish was replaced with fresh medium. UPEC132 was grown overnigh

19、t on LB agar andthen suspended gently in PBS (0.01M, pH7.4) at a concentration of 108 CFU/ml. Two hundred microliters ofUPEC132 suspension were added into the dishes. The uninfected cells were used as a negative control. Thecultures were placed in air with 5% CO2 at 37°C and observed with an in

20、verted microscope every 15 min. Whenthe cell morphology of experiment group showed significant changes, the cells were washed three times withPBS, fixed with 4% paraformaldehyde, and stained with Giemsa stain. The assay was run in triplicate andrepeated three times. The adherence rate was determined

21、 by randomly counting 100 cells, and the result wascalculated as following formula: the number of cells bound with bacteria/ the number of cells counted 100%. 2.4 Confocal microscopy observation for bacterial invasion Plasmid pSELECT-GFPzeo-mcs carrying GFP gene was transformed into UPEC132 using st

22、andard methods asdescribed. The recombinant UPEC132 expressing GFP was identified by a fluorescence microscope anddesignated UPEC132/pSELECT-GFP. EJ cells were seeded onto glass coverslips in 6-well plates (Corning, NY,USA) and grown to confluence. UPEC132/pSELECT-GFP was treated as UPEC132 describe

23、d in adherenceassays. Twenty microliters of bacterial suspension were added into the wells. Following 1h incubation,coverslips were gently washed three times in PBS, then visualized and photographed using an OlympusFluoView FV300 inverted confocal laser scanning microscope. Images were captured with

24、 0.1m step to obtainintracellular sections of bright field and fluorescent field. Then both sections of bright and fluorescent field weremerged by using Olympus FV300 software in order to show whether bacteria were located intracellularly. 2.5 Analysis of mRNA expression using cDNA microarrays EJ ce

25、lls were infected with UPEC132 as described above, and uninfected cultures were used as negative control. After 1 h incubation, total RNA was extracted from the cells with the Trizol reagent (Invitrogen, CA, USA)according to the manufacturers instructions, and RNA integrity was confirmed by gel elec

26、trophoresis. Themicroarray analysis was performed by CapitalBio Corporation (Beijing, China) using a human genome 70-meroligonucleotide microarray (QIAGEN, Hilden,Germany) consisted of 22 000 distinct cDNAs. The array alsoincluded cDNAs of housekeeping genes, such as -actin and glyceraldehyde-3-phos

27、phate dehydrogenase(GAPDH), to serve as internal controls. Cy5-dCTP or Cy3-dCTP was incorporated when 5g total RNA wasreverse-transcribed into cDNA using a cDNA synthesis system (TaKaRa, Dalian, China). The cDNA from thecells co-cultured with UPEC132 were incorporated with Cy5, while the cDNA from t

28、he uninfected cells wereincorporated with Cy3. Labeled test and control samples were mixed in 80l hybridization buffer (3SSC, 0.2%sodium dodecyl sulfate, 25% formamide, and 5Denhardts solution). DNA in hybridization solution wasdenatured at 95°C for 3 min before loading onto a microarray. The a

29、rray was hybridized at 42°C overnight andwashed with two consecutive washing solutions (0.2% sodium dodecyl sulfate, 2SSC at 42°C for 5 min, and0.2SSC for 5 min at room temperature). Arrays were scanned with a ScanArray Express scanner (ParckardBioscience, Kanata, OT), and the obtained ima

30、ges were analyzed with GenePix Pro 4.0 (Axon Instruments,FosterCity, CA). A Cy5: Cy3 ratio between 2 and 0.5 indicated only small mRNA expression differences andwas of no significance. A ratio of more than 2 or less than 0.5 indicated significant mRNA expressiondifferences. 2.6 Semi-quantitative RT-PCR RT-PCR analysis was performed on the same set of RNAs used for the microarray analysis.The total RNA wasreverse-transcribed into cDNA using P