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  本文選題：G-CSF動員　切入點：MDC　出處：《安徽醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： G-CSF動員的供體外周血單個核細胞細胞貼壁后,在含IL-4和GM-CSF的無血清培養(yǎng)基中誘導培養(yǎng)7d,再用IL-10、ATG和TNFa進一步分組誘導2d。收集上清液及細胞,分別用流式細胞術、ELISA、抗原吞噬實驗和混合淋巴細胞反應(MLR)測定各組DC的HLA-DR、CD1a、CD11c、CD40、CD80分子表達,IL-12(P70)分泌,抗原吞噬功能以及同種異體反應刺激功能。 結果 從G-CSF動員的供體外周血中能誘導產生未成熟DC(iDC),CD1a~+DC細胞產率為1.62±0.57×10~4/2×10~6PBMCs。iDC經TNFa、IL-10/TNFa和ATG/TNFa分組誘導后:1.與iDC相比,TNFa刺激后,HLA-DR、CD11c、CD40、CD80的表達均明顯增高,IL-12分泌增加,同種異體反應刺激能力增強。2.與TNFa刺激組相比,IL-10/TNFa組CD1a表達增高,對應的其他分子表達均明顯減弱,IL-12分泌及同種異體反應刺激能力明顯減低。3.與TNFa刺激組相比,ATG/TNFa組HLA-DR、CD11c、CD40、CD80表達均明顯減弱,IL-12分泌和同種異體反應刺激能力明顯減低。 4.ATG/TNFa組與IL-10/TNFa組相比,CD1a表達減弱,CD11c、CD80表達明顯增高,同種異體反應刺激能力增強。5.iDC經TNFa誘導成熟后,吞噬抗原的能力明顯減弱。6.從G-CSF動員的供體PBMCs中誘導產生的iDC,其HLA-DR、CD11c表達明顯低于未經動員的健康對照來源的iDC。TNFa刺激后,CD11c、CD40、CD80表達、IL-12分泌和同種異體反應刺激能力均明顯低于健康對照。 結論 1、從G-CSF動員的供體外周血單個核細胞中可以誘導產生iDC,TNF-a能誘導其分化成熟,IL-10、ATG誘導可使其獲得致耐受的特性。 2、與健康獻血員相比,G-CSF動員的供體外周血單個核細胞來源的DC表型相對幼稚,同種異體反應刺激能力較弱。 3、盡管從G-CSF動員的供體PBMC中誘導產生CD1a~+DC的效率不及未經動員的健康獻血員高,但因從動員的供體外周血中可以采集到大量的PBMCs,因此,G-CSF動員的外周血有望成為不同DC的重要來源途徑。 二、G-CSF動員的供體不同DC亞群免疫生物學特性的研究 實驗方法 用免疫磁珠分離髓細胞來源的DC(MDC)和漿細胞來源的DC(PDC),并分別用TNFa、CpG_(2006)OND誘導為成熟MDC(mMDC)和成熟PDC(mPDC)。具體的研究方法如下:1.流式細胞術測定各DC亞群的表型;2.MLR測定不同DC亞群同種異體反應刺激能力;3.ELISA測定MLR培養(yǎng)上清液中INF-γ、IL-10的含量,細胞內流式術測定MLR后CD4~+T細胞的。INF-γ和IL-4分泌水平;4.流式細胞術和RT-PCR測定MLR后CD4~+CD25~(high)T細胞比例和Foxp3mRNA的表達。 結果 1.分選的MDC純度＞96%,高表達HLA-DR、CD11c、CD33,中度表達CD4,極低表達CD40、CD45RA、CD80,經TNFa誘導成熟后CD40、CD80表達上調。PDC純度＞95%,高表達HLA-DR、CD4、CD45RA,極低表達CD11c、CD33、CD40、CD80,經CpG2006OND誘導成熟后CD40、CD80表達上調。 2.MDC特別是mMDC具有很強的刺激T淋巴細胞增殖能力,但PDC、mPDC同種異體反應刺激能力很弱。 3.mMDC刺激后,T細胞分泌INF-γ水平明顯高于MDC和PDC刺激。PDC特別是mPDC刺激后,IL-10明顯增高。 4.MDC和mMDC刺激后,CD4~+T細胞胞漿內IFN-γ的表達均明顯高于對照組和PDC刺激組,mMDC刺激組的表達最高。所有刺激組CD4~+T細胞內IL-4的表達無明顯差異。 5.MDC和mMDC對CD4~+CD25~(high)Treg無明顯影響,PDC則上調Treg,mPDC的作用更為明顯; 6.Foxp3mRNA的表達在各組間無明顯的差別。 結論 1.采集的供體外周血中兩類DC亞群雖然HLA-DR高表達,但仍處于非成熟狀態(tài),在合適的條件下分化成熟;無論MDC成熟與否均表現出很強的刺激T細胞增殖能力,可以促進使T細胞分泌IFN-γ增加,其分泌的增加可能是促進向Th1極化的結果。 2.PDC可能并不像MDC一樣有效地捕獲、處理和負載抗原到MHC分子上,因此呈遞抗原效率低,表現出對T細胞的弱刺激增殖特性;PDC雖然也可以促進T細胞分泌IFN-γ的分泌,但似乎并非是通過Th1細胞分泌:PDC并不能促進Th2類因子IL-4的分泌,對Th2的極化作用可能表現在IL-10的分泌上。 3.PDC有誘導CD4~+CD25~(high)Treg的作用。
[Abstract]:G-CSF mobilized donor peripheral blood mononuclear cells to adherent cells in serum-free medium, cultured 7d, and then IL-10 containing IL-4 and GM-CSF, ATG and TNFa further group induced 2D. supernatant was collected and cells respectively by flow cytometry, ELISA, antigen phagocytosis test and mixed lymphocyte reaction (MLR) determination of serum DC HLA-DR, CD1a, CD11c, CD40, expression of CD80, IL-12 (P70) secretion and phagocytosis of antigen and allogeneic stimulation.
Result
From G-CSF mobilized peripheral blood donor can induce immature DC (iDC), CD1a~+DC cell yield was 1.62 + 0.57 * 10~4/2 * 10~6PBMCs.iDC by TNFa, IL-10/TNFa and ATG/TNFa groups after induction: 1. compared with iDC, TNFa, HLA-DR, CD11c after stimulation, CD40 CD80 expression was significantly increased, the increase of IL-12 the secretion of allogeneic stimulation enhance the ability of.2. stimulation compared with TNFa group, IL-10/TNFa group increased the expression of CD1a, the expression of the corresponding other molecules were significantly decreased, IL-12 secretion and decreased ability to stimulate alloreactive.3. stimulation compared with TNFa group, ATG/TNFa group, HLA-DR, CD11c, CD40, CD80 expression significantly decreased, and the secretion of IL-12 allogeneic stimulatory capacity decreased significantly.
Compared with 4.ATG/TNFa group and IL-10/TNFa group, the reduced expression of CD1a, CD11c, CD80 expression was significantly increased, allogeneic stimulation enhanced the ability of.5.iDC induced by TNFa after maturation, phagocytosis of antigen induced by.6. significantly decreased from G-CSF mobilized donor PBMCs iDC, the HLA-DR, CD11c expression was significantly lower than that in the mobilization of healthy control source after iDC.TNFa stimulation, CD11c, CD40, CD80 expression, IL-12 secretion and stimulate alloreactive capacity were significantly lower than that of healthy controls.
conclusion
1, iDC can be induced from peripheral blood mononuclear cells mobilized by G-CSF, and TNF-a can induce differentiation and maturation. IL-10 and ATG induce it to achieve tolerance.
2, compared with the healthy blood donors, the DC phenotype of G-CSF mobilized donor peripheral blood mononuclear cells is relatively immature, and the allogenic reaction is weak.
3, although the efficiency of inducing CD1a~+DC from donor PBMC mobilized by G-CSF is higher than that of unmobilized healthy blood donors, a large number of PBMCs can be collected from mobilized donor peripheral blood. Therefore, peripheral blood mobilized by G-CSF is expected to become an important source of different DC.
Study on the immunological biological characteristics of two, G-CSF mobilized donor different DC subgroups
Experimental method
The separation of myeloid derived by immunomagnetic beads of DC (MDC) and plasma cells derived DC (PDC), were treated with TNFa, CpG_ (2006) OND MDC (mMDC) induced into mature and mature PDC (mPDC). The specific research methods are as follows: 1. phenotypes were detected by flow cytometry in various DC subsets 2.MLR; Determination of different subsets of DC allogeneic stimulation ability; 3.ELISA determination of MLR in supernatant of INF- gamma, IL-10 content, cell flow cytometry determination of MLR of CD4~+T cells after.INF- gamma and IL-4 secretion; 4. flow cytometry and RT-PCR assay after MLR CD4~+CD25~ (high) T cells and expression than cases Foxp3mRNA.
Result
1. sorting MDC purity is more than 96%, the high expression of HLA-DR, CD11c, CD33, moderate expression of CD4, low expression of CD40, CD45RA, CD80, induced by TNFa after mature CD40, upregulation of CD80.PDC purity is more than 95%, the high expression of HLA-DR, CD4, CD45RA, low expression of CD11c, CD33, CD40, CD80, the CpG2006OND induced mature CD40, upregulate the expression of CD80.
2.MDC, especially mMDC, has a strong ability to stimulate T lymphocyte proliferation, but PDC, mPDC allograft response is very weak.
After 3.mMDC stimulation, the level of INF- gamma secreted by T cells was significantly higher than that of MDC and PDC stimulated.PDC, especially after mPDC stimulation, and IL-10 was significantly increased.
After stimulation with 4.MDC and mMDC, the expression of IFN- gamma in CD4~+T cells was significantly higher than that in the control group and PDC stimulation group. The expression of mMDC in the mMDC stimulation group was the highest. There was no significant difference in IL-4 expression in all stimulation group CD4~+T cells.
5.MDC and mMDC had no obvious effect on CD4~+CD25~ (high) Treg, while PDC increased Treg, and mPDC had more obvious effect.
There was no significant difference in the expression of 6.Foxp3mRNA between the groups.
conclusion
The two class of 1. donor peripheral blood DC subsets while high expression of HLA-DR, but is still in the immature state, under the suitable conditions of differentiation and maturation of MDC; whether mature or not showed a strong stimulation of the proliferation ability of T cells, T cells can promote the secretion of IFN- increased the secretion increase may promote the result of Th1 polarization.
Unlike MDC and 2.PDC could effectively capture, processing and antigen to the MHC molecule, so the antigen presentation efficiency is low, showing the proliferation characteristics of weak stimulation on T secretion of PDC cells; although also can promote IFN- secretion of T cells, but it was not secreted by Th1 cells: PDC did not promote the secretion of Th2 factor IL-4, the polarization effect of Th2 might reflect the production of IL-10.
3.PDC has the effect of inducing CD4~+CD25~ (high) Treg.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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