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Interaction between Tfh Cells,B Cells and HepG2.2.15Cells

Author: LiangWen
Tutor: RenHong
School: Chongqing Medical University
Course: Internal Medicine
Keywords: immunomagnetic bead B lymphocytes Tfh cell Co-culture system Costimulatory molecules
CLC: R392
Type: Master's thesis
Year: 2012
Downloads: 132
Quote: 0
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ObjectiveTake the Cell membrane receptor CXCR5as the common antigen marks and applyimmunomagnetic beads sorting method to establish a common training system whichis made by Tfh cells and B cells. Observe the changes of cytokines between thetraining system and human hepatocellular carcinoma cell lines, which should be basedon the establishing the common system successfully.All of my study is to lay afoundation to further the study of the relationship among the In vitro study of the cell.Methods1.The research object:15patients who suffer splenectomy in patients withchronic hepatitis B liver cirrhosis are needed (male8and female7).2. Flow cytometric detection: Peripheral blood20ml are needed before operationof splenectomy in patients with chronic hepatitis B liver cirrhosis. During theoperation, take about10g sample to analysis of the obtained lymphocyte, relevantmarked antibody and Cell phenotype originated from flow cytometric detection inPeripheral blood and Tfh cells from spleen.3. Use immunomagnetic beads method to establish a common training system forTfh cells and B cells: adopt the gradient centrifugation to separate15patients ofchronic hepatitis B virus infection and spleen mononuclear cell (PBMC). Set the Cellmembrane receptor as the antigen marker. Adopt the Immunomagnetic beads sortingmethod to separate the CXCR5+cells. Test the cell purity and composition ofthe CXCR5+cells by flow cytometry.4.Mixed cultivation:Put the CXCR5+cells and human liver cell lines HepG2.2.15cells in vitro mixed cultivating1week.5. Flow cytometric detection: After the CXCR5+cells and human liver cell linesHepG2.2.15cells in vitro have been mixed cultivated for one week, use the flowcytometry to detect the Tfh cell phenotype change.6. Mixed culture supernatant detection: After one week of mixed cultivating,Apply ELISA to test the culture supernatant HBeAb.7. Mixed culture supernatant of HBV DNA determination: After the treatment ofthe collection of the supernatant by DNA extract, use the PCR amplification, followingoperation procedures of HBV DNA fluorescent quantitative PCR detecting reagentbox.8. Mixed cultured cells CXCR5mRNA determination:(1) the extraction totalRNA: According to the high purity of total RNA Extraction Kit instructions.(2)Synthesis of cDNA: With every10ul reaction system of400ng RNA by reversetranscription kit instruction synthesis.(3) PCR amplification: CXCR5primer:F:5-CTCCTCTCCATCCACATCACCT-3R:5-CGTTTCTGCTTGGTTCTCTTGG-3.Reaction conditions95℃30S;95℃5S,60℃31S totally with40cycle. Use the2-△△CTmethods for the analysis of gene expression levels and B-actin as the internal control.9. Statistical methods: use SPSS17.0software to analyze and use the t test andanalysis of variance. P <0.05for differences has Statistical significance.Results1. Peripheral blood as a source of Tfh cells and spleen derived Tfh cellphenotype exist difference in the same patients. The expression of Peripheral bloodTfh cells from CCR7, PD-1,PDL-1is higher than spleen Tfh cells so it has Statisticalsignificance(P<0.05). While the expression of ICOS is lower than splenic Tfh cells, sothe difference was statistical significance.(P<0.05).2.The secretion of cytokines IFN-(r8.32±1.21)of Peripheral blood as a source ofTfh cells from the same patients is higher than the secretion of cytokines IFN-r(3.83±0.32)of Spleen derived Tfh obviously, which has statistical significance.3.Application of magnetic bead sorting method can successfully obtained CXCR5+cell: Use flow cytometry to identify purity and composition of the cell.4. After the CXCR5+cells and human hepatoma cell line HepG2.2.15cellshave been mixed cultured in vitro for1week, use flow cytometry to test Tfh cellphenotypic change, finding PD1,PDL-1,ICOS are lower than the culture before,which has statistical significance.5. After the supernatant of HBeAb have been mixed cultured for one week, thequantitative test results are: Experimental group HBeAb(0.768±0.094) are higherthan blank control group HBeAb(0.478±0.088), which has statistical significance.6.Mixed culture supernatant of HBV-DNA quantitative test results: Experimentalgroup HBV-DNA is obviously higher than blank control group. The inhibition rate ofHBV-DNA was33%.7.After the CXCR5+cells and human hepatoma cell line HepG2.2.15cells havebeen mixed cultured in vitro for1week, Detect the CXCR5+cells and CXCR5mRNA expression finding there is no significantly difference between experimentalgroup and blank control group.Conclusion1. There are differences between the Spleen derived Tfh cells and peripheralblood derived Tfh cell phenotypic. After CXCR5+cells and human hepatoma cellline HepG2.2.15cells are in a mixed culture, Spleen derived Tfh cell phenotype hasthe Peripheral blood Tfh cell phenotype change trendency.2. The use of indirect immunomagnetic separation method can establish Coculture system of CXCR5+B lymphocytes and Tfh cells In order to lay a foundation tofurther study the cell interaction.3. CXCR5+cells have inhibition effect on HBV-DNA replication and Theinhibition rate of HBV-DNA was33%. They also inhibition effect on HBeAb.According to the amplification of CXCR5, there is no obvious difference betweenexperimental group and blank control group, which shows that the influence ofCXCR5+cells on HBV-DNA replication is not achieved by high expression ofCXCR5. That is to say, the specific action mechanism remains to be further studied.

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