G-CSF动员的供体外周血树突状细胞免疫生物学特性的研究

发布时间：2018-03-20 23:36

本文选题：G-CSF动员　切入点：MDC　出处：《安徽医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： G-CSF动员的供体外周血单个核细胞细胞贴壁后,在含IL-4和GM-CSF的无血清培养基中诱导培养7d,再用IL-10、ATG和TNFa进一步分组诱导2d。收集上清液及细胞,分别用流式细胞术、ELISA、抗原吞噬实验和混合淋巴细胞反应(MLR)测定各组DC的HLA-DR、CD1a、CD11c、CD40、CD80分子表达,IL-12(P70)分泌,抗原吞噬功能以及同种异体反应刺激功能。结果从G-CSF动员的供体外周血中能诱导产生未成熟DC(iDC),CD1a~+DC细胞产率为1.62±0.57×10~4/2×10~6PBMCs。iDC经TNFa、IL-10/TNFa和ATG/TNFa分组诱导后:1.与iDC相比,TNFa刺激后,HLA-DR、CD11c、CD40、CD80的表达均明显增高,IL-12分泌增加,同种异体反应刺激能力增强。2.与TNFa刺激组相比,IL-10/TNFa组CD1a表达增高,对应的其他分子表达均明显减弱,IL-12分泌及同种异体反应刺激能力明显减低。3.与TNFa刺激组相比,ATG/TNFa组HLA-DR、CD11c、CD40、CD80表达均明显减弱,IL-12分泌和同种异体反应刺激能力明显减低。 4.ATG/TNFa组与IL-10/TNFa组相比,CD1a表达减弱,CD11c、CD80表达明显增高,同种异体反应刺激能力增强。5.iDC经TNFa诱导成熟后,吞噬抗原的能力明显减弱。6.从G-CSF动员的供体PBMCs中诱导产生的iDC,其HLA-DR、CD11c表达明显低于未经动员的健康对照来源的iDC。TNFa刺激后,CD11c、CD40、CD80表达、IL-12分泌和同种异体反应刺激能力均明显低于健康对照。结论 1、从G-CSF动员的供体外周血单个核细胞中可以诱导产生iDC,TNF-a能诱导其分化成熟,IL-10、ATG诱导可使其获得致耐受的特性。 2、与健康献血员相比,G-CSF动员的供体外周血单个核细胞来源的DC表型相对幼稚,同种异体反应刺激能力较弱。 3、尽管从G-CSF动员的供体PBMC中诱导产生CD1a~+DC的效率不及未经动员的健康献血员高,但因从动员的供体外周血中可以采集到大量的PBMCs,因此,G-CSF动员的外周血有望成为不同DC的重要来源途径。二、G-CSF动员的供体不同DC亚群免疫生物学特性的研究实验方法用免疫磁珠分离髓细胞来源的DC(MDC)和浆细胞来源的DC(PDC),并分别用TNFa、CpG_(2006)OND诱导为成熟MDC(mMDC)和成熟PDC(mPDC)。具体的研究方法如下:1.流式细胞术测定各DC亚群的表型;2.MLR测定不同DC亚群同种异体反应刺激能力;3.ELISA测定MLR培养上清液中INF-γ、IL-10的含量,细胞内流式术测定MLR后CD4~+T细胞的。INF-γ和IL-4分泌水平;4.流式细胞术和RT-PCR测定MLR后CD4~+CD25~(high)T细胞比例和Foxp3mRNA的表达。结果 1.分选的MDC纯度＞96%,高表达HLA-DR、CD11c、CD33,中度表达CD4,极低表达CD40、CD45RA、CD80,经TNFa诱导成熟后CD40、CD80表达上调。PDC纯度＞95%,高表达HLA-DR、CD4、CD45RA,极低表达CD11c、CD33、CD40、CD80,经CpG2006OND诱导成熟后CD40、CD80表达上调。 2.MDC特别是mMDC具有很强的刺激T淋巴细胞增殖能力,但PDC、mPDC同种异体反应刺激能力很弱。 3.mMDC刺激后,T细胞分泌INF-γ水平明显高于MDC和PDC刺激。PDC特别是mPDC刺激后,IL-10明显增高。 4.MDC和mMDC刺激后,CD4~+T细胞胞浆内IFN-γ的表达均明显高于对照组和PDC刺激组,mMDC刺激组的表达最高。所有刺激组CD4~+T细胞内IL-4的表达无明显差异。 5.MDC和mMDC对CD4~+CD25~(high)Treg无明显影响,PDC则上调Treg,mPDC的作用更为明显; 6.Foxp3mRNA的表达在各组间无明显的差别。结论 1.采集的供体外周血中两类DC亚群虽然HLA-DR高表达,但仍处于非成熟状态,在合适的条件下分化成熟;无论MDC成熟与否均表现出很强的刺激T细胞增殖能力,可以促进使T细胞分泌IFN-γ增加,其分泌的增加可能是促进向Th1极化的结果。 2.PDC可能并不像MDC一样有效地捕获、处理和负载抗原到MHC分子上,因此呈递抗原效率低,表现出对T细胞的弱刺激增殖特性;PDC虽然也可以促进T细胞分泌IFN-γ的分泌,但似乎并非是通过Th1细胞分泌:PDC并不能促进Th2类因子IL-4的分泌,对Th2的极化作用可能表现在IL-10的分泌上。 3.PDC有诱导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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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