The Study Of The Dendritic Cells' Function In Inflammatory Bowel Disease

Strong immunoreaction is osculation related with IBD, the DC can processing antigen and adjust the immunoreaction. DC is the beginning actor of the immunoreaction, and induce the immunity response. The research on the DC not only help us realize the adjust mechanism of the body's immunity response, but also balance the body's immunity response by adjust the DC function.The first part: The study on establishment of the mouse colitis modelMethods:Statistics analysis: one-way ANOVA and multiple comparisons with Dunnett way by the spss 10.0 software, when P＜0.05 the difference is significant.Totally 50 female Balb/c mice were divided into 5 groups by chanced, 10 mice each one groups. The first group was control group was instilled the 30% ethanol. The others groups were instilled by 25mg/kg-150mg/kg TNBS dissolved in 30% ethanol into mice' colon to induce distal colitis. One mice was killed in 3, 7, 21, 28 day every group. The disease activity index (DAI), macroscopical, histological change of the colon and myeloperoxidase(MPO) activity of the mucosa were evaluated.Result:Diarrhea, blood stool, hyperemia and enema of the control and the 25mg/kg group was healed in one week. The 50mg/kg-150mg/kg groups was appeared of diarrhea, blood stool, ulcer. Histological change is that colon incrassation, hyperemia, enema, lymphocytes and plasma cells became predominant. The tissue MPO activity increased. The phenomenon lased for three weeks, and was healed after three weeks.Conclusions:TNBS induced mice colitis is an ideal model of IBD, and may serve as a tool for investigation of the pathogenesis and pharmaceutical effect of IBD.The second part: study on the surface marker analysis and the IL-2 express of dendritic cell in colitis of mice.Method:Statistics analysis: independent-samples t test by the spss 10.0 software, when P＜0.05 the difference is significant.Isolation of mouse spleen dendritic cells1. Study on separation of dendritic cells from mouse spleen1.1 Sample preparation1.1.1 Place isolation spleen in a 6cm petri-dish with sufficient Collagenase D solution to completely cover the bottom of the dish(5mL/spleen).1.1.2 Inject mouse spleen with 500uL of Collagenase D solution per spleen using a 1mL syringe and a 25G needle, then cut the tissue into smaller pieces using sharp scissors.1.1.3 Incubate the spleen pieces in Collangenase D solution for 30 minutes at 37℃1.1.4 Pass the whole material, i. e. remaining fragments and Collagenase D-released cells, gently through a 70 um cell strainer using a plunger.1.1.5 Collect all cells in a 15mL tube and wash the cells by adding buffer to a final volume of 14mL.1.1.6 Proceed to magnetic labeling.1.2 Magnetic labeling1.2.1. Determined cell number.1.2.2. Centrifuge cell suspension at 1000rpm for 10 minutes. Pipette off supernatant completely. 1.2.3. Resuspend cell pellet in 400uL of buffer per 10⁸ total cells.1.2.4. Add 100uL of CDllc (N418) Microbeads per 10⁸ total cells.1.2.5. Mix well and incubate foe 15 minutes at 4-8℃.1.2.6. Wash cells by adding 1-2mL of buffer per 10⁸ cells and centrifuge at 200^*g for 10 minutes, pipette off supernatant completely.1.2.7. Resuspend up to 10⁸ cells in 500uL of buffer.1.2.8. Proceed to magnetic separation.1.3 Magnetic separationChoose an miniMACS Column and miniMACS Separator.1.3.1 Place column in the magnetic field of a suitable1.3.2 Prepare column by rinsing with 500uL of buffer.1.3.3 Apply cell suspension onto column.1.3.4 Collect unabled cells which pass through and wash column with 1500uL of buffer. Perform washing steps by adding buffer three times, each time once the column reservoir is empty. Collect total effluent. This is the unlabeled cell fraction.1.3.5 Remove column from the separator and place it on a suitable collection tube.1.3.6 Pipette appropriate amount of buffer onto the column. Iimmediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column.1.3.7 To increase the purity of the enriched CD11c cells, repeat the magnetic separation procedure as described in steps 1-6 by using a new column.2. Study on separation of dendritic cells from mouse spleen by half conglutination.Kill the mouse by disjoint the neck vertebra, dip in the 70% ethanol for 2 minutes. Take out the spleen by without asepsis. Place isolation spleen in a 6cm petri-dish with sufficient Hank's solution. Cut the spleen into pieces by sharp scissor, and pass the whole material gently through a 70um cell strainer using a plunger. Collect all cells in a 15mL tube and add the red blood cell lysis buffer for 5 minutes at 4℃. Centrifuge cell suspension at 1000rpm for 10 minutes. Resuspend cell of 5%BSA-RPMI culture solution per 10⁷ total cells. Throw the metrizamide gradient and transfer 2.5mL into a sterile 15mL tube. Overlay cells slowly onto the gradient. It is important not to disturb the gradient. Spin the gradient for 10 minutes at 1500rpm at room temperature with no break on the centrifuge. Collect cells from the interface using a 5mL pipet and wish them once with Hank's solution. Resuspend cells in 1mL of. Place the cells into 10cm plate and place the plate in 5%CO₂ culture box for 2 hours at 37℃. Wish off the not conglutination cells by Hank's solution, add 5%BSA-RPMI into the rest cells and place it in 5%CO₂ culture box for 16 hours at 37℃. The dendritic cells become not conglutination, wave the plate softly and pipette the suspension.Labeling with anti-CD11c antibody on the dendritic cell surface isolated by two ways partly and analysis the purity of the antibody on the Flow Cytometry.Study on analysis surface marker of dendritic cells1. After isolate the dendritic cells from mouse spleen, the control group and the test group are partly labeled with Fitc-CD80, PE-CD86, PE-CD83, then analysis on the flow cytometry.2. Make the mouse spleen into the mononuclearcells, rid off the red blood cells, double labeled with CD11c and Fitc-CD80, PE-CD86, PE-CD83, then analysis on the flow cytometry.Flow Cytometry: pipette about 10⁵ total cells in the needle bottom tube. Partly adding antibody into the tube, incubate for 15-30min at 4℃. Wish cells once using PBS solution and Centrifuge cell suspension at 1000rpm for 5min. add appropriate PBS solution, proceed analysis on FCM.Study on analysis expression of interleukin-2 by RT-PCR.Total RNA was isolated from colon tissue and cells. The mRNA was separated. The interleukin-2 primer pair was designed as: 5'-a a c c t g a a a c t c c c c a g g a t-3', 3'-t c c a c c a c a g t t g c t g a c t c -5, product length is 254 base pairs. GAPDH as an internal standard template and its primer pair is: 5'-a g a c a g c c g c a t c t t c t t g t-3', 3' -t c t c c a t g g t g g t g a a g a c a-5' product length is 361 base pairs. Interleukin-2 and GAPDH cDNA was generated by RT-PCR amplificationResult:1. The purity of CD11c antibody is 45.0% by using the half of conglutination. But the purity of that using the MicroBeads is 66.1%, when use the more miniMACS Column the purity is up to 99%. The p purity by using MicroBeads is higher than using the half of conglutination.2. The first method result: the percent of control group Fitc-CD80,PE-CD83,PE-CD86 is 7.6333, 6.8100, 6.3600, the test group is 27.0767, 16.0900, 27.2500. By the paired samples t examilation, the test group is higher than the control group.The second result: the percent of control group Fitc-CD80, PE-CD86, PE-CD83 is 9.800, 7.3250, 11.475, the test group is 14.625, 11.7500, 14.1500. By the paired samples t examination the test group is higher than the control group.3. The IL-2 expression of test DC, lymphocyte and colon tissue is higher than the control group's.Conclusion:1. Using the MicroBeads isolation the dendritic cells fron mouse spleen is better than using the half of conglutination, when using the more miniMACS Column, the content is up to 99%. Using the MicroBeads isolation the dendritic cells is the best way.2. The study discovered that the surface marker of dendritic cells in the mouse colitis model is higher than the healthy mouse. CD80, CD86, CD83 is high register that the dendritic cells are mature cells, and which is exert the function of present antigen.3. The research discovery that in colitis the expression of IL-2 is higher than the normal. In DC the expression of IL-2 is high. DC excrete IL-2, and lymphocyte also excrete IL-2. IL-2 is the devote inflammatory factor of Th1, DC and lymphocyte interacter, blow up the immunoreaction, and reduce the disease at last.