【作者】 徐英萍；

【导师】 王家富； 李伟；

【作者基本信息】 泰山医学院 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:1.拟建立稳定的小鼠子宫蜕膜基质细胞(decidual stromal cell, DSC)培养方法,观察DSC形态特征和生物学特性。2.在体外建立稳定的从小鼠骨髓来源的单核细胞诱导成熟树突状细胞(dendritic cell, DC)的培养体系,观察DC形态特征和生物学特性。3.建立细胞共培养模型,初步探讨小鼠蜕膜基质细胞对成熟DC增殖的影响,为进一步研究DC在诱导母胎免疫耐受中的作用打下良好的基础。方法:1.选用孕龄6-8d的C57BL/6孕鼠蜕膜组织,采用胰蛋白酶、胶原酶、透明质酸酶混合消化,进行蜕膜基质细胞体外培养。倒置显微镜观察细胞形态学变化;催乳素(prolactin, PRL)免疫组织化学染色进行细胞鉴定;RT-PCR检测DSC表达的细胞因子和趋化因子。2.应用粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和白细胞介素-4(interleukin 4,IL-4)诱导培养小鼠骨髓细胞,相差显微镜观察形态学变化;FACS检测细胞表面分子;3H-TdR掺入法检测DC刺激同种异体或同体T细胞增殖能力;ELISA检测其分泌的细胞因子。3.采用小鼠子宫DSC和成熟DC共培养技术,建立细胞共培养模型,观察DSC对DC增殖的影响。结果:1.小鼠子宫DSC在接种24h后大部分已贴壁,贴壁生长的细胞成纤维母细胞状, 5-7d后细胞融合成单层。每4-5d传代1次,传代的细胞形态无明显变化,而传代培养极性渐不明显。PRL免疫组化显示体外培养传代后95%阳性细胞为蜕膜基质细胞,细胞大小形状均一,染色特点为胞质内可见细小的棕色颗粒,且越近胞核染色越深。RT- PCR检测DSC表达GM-CSF、血管内皮生长因子(vascular endothelial cell growth factor,VEGF)、白介素-2(interleukin 2, IL-2)、白介素-10(interleukin-10, IL-10)等细胞因子;表达单核细胞趋化蛋白-2(Monocyte chemotactic protein-2, MCP-2)、单核细胞趋化蛋白-3(Monocyte chemotactic protein-3, MCP-3)、胸腺活化相关的趋化性因子(Thymus activation-related chemokine,TRAC)等趋化因子。2.用GM-CSF+ IL-4细胞因子联合培养3d后,有细胞集落形成;培养第5d开始,一部分细胞从集落脱落,悬浮于培养基中,呈现树枝状不规则变化;脂多糖(lipopolysaccharide,LPS)诱导后,成簇的细胞散开,大量的细胞脱落,多数为圆形或不规则形,表面有丰富的伸长毛刺的低密度大细胞。成熟DC高表达DC相对特异性标志CD11c、共刺激分子CD80、CD86、MHCⅡ类分子Ia和Ⅰ类分子H-2Kb。DC具有强烈的激活T细胞增殖的能力;1000个DC,即可有效激发T细胞增殖。LPS刺激成熟8d DC与未经LPS刺激5d DC,其刺激同种异体或同体T细胞增殖能力明显增高,结果比较差异有统计学意义(P<0.05)。并且在各组中随着DC:T比例增加,其刺激T细胞增殖能力增强,结果比较差异有统计学意义(P<0.05)。经LPS刺激的成熟DC分泌细胞因子IL-12p70、IL-6、TNFa和IL-1b的水平明显升高,与未经LPS刺激的PBS组结果比较差异有统计学意义(P<0.05)。3.成熟DC能在DSC单层上继续生长,没有随着培养时间的延长而走向自然凋亡,第15d镜下观察形成细胞集落,发生了克隆性增殖。结论:1.建立的混合酶消化全组分蜕膜的方法,简化了DSC的提纯程序,能获得高纯度的合乎实验要求的DSC,具有经济、实用、方便、可靠的特点。2.细胞因子组合GM-CSF+IL-4+LPS在体外具有诱导小鼠骨髓来源的单核细胞分化为成熟DC的作用,我们建立的体外诱导小鼠骨髓单核细胞的方法可以获得形态、表型和功能都非常典型的成熟DC,方法简便。3.小鼠子宫DSC组成的微环境能逆转成熟DC的自然凋亡,使其重新获得增殖的能力。更多还原

【Abstract】 【Objective】1.To develop a simple and satisfactory method to culture mice decidual stromal cell(DSC). The construction of a co-coltured system of decidual cells will lay a good foundation to study pathological pregnancy.2. To establish method of expanding dendritic cells from mice bone marrow in vitro. The cells morphological changes were observed by inverted microscope . Finally,we studied on its Biological Characteristic and Identification the cells.3. A Preliminary Investigation on effect of DC proliferation in DSC/mDC co-culture system.【Methods】1.Decidual tissues of pregnancy on days 6 through 8 from C57BL/6 mice were digested with trypsin,hyalace and collagenase,then cultured of composition of decidual cells. Erythrocytes,lymphocytes and non-adherent components were removed by changing medium to overnight primary cells.After being passaged for three times,the DSCwere isolated and purified.After purification,the growth features of the cultured cells were observed and finally idencified by immunohistochemistry using prolaction(PRL)kit. Cytokine and chemokine secreted by DSC was tested by RT-PCR.2. Cells isolated from mice bone marrow were cultured in GM-CSF and IL-4, morphological change was observed by optical phase contrast microscopy, T cell proliferation was detected by 3H-TdR incorporting into assay, and surface molecular was detected by FACS, and cytokines were assayed by ELISA.3.At last,we sorted mature DC from murine bone marrow,and culured them with DSC for at least 5 days.Then we observed proliferated changes of DC. 【Results】1. Almost the cultured DSC were adhered Twenty four hours after implantion,and the adhered cells showed the shape of fibroblast.Monolayer cells were formed on days 5 through 7.Primary cultured DSC were amplified for several weeks,passaged one time on days 4 through 5.The shape of passaged cells was not changed,but the polarity of passaged DSC was not obvious gradually.The immunohistochemistry revealed that PRLpositivity was 95%,the shape and size of cells were approximately similar.There were fine brown-yellow particles in the cytoplasm and the deeper color was close to nucleus.Some cytokines and chemkines were assayed by semi-quantitative RT-PCR.It implied that some stimulating factors have been produced by the stroma.2.Bone marrow mononeuclear cells,prepared from the mouse femurs by depletion of red cells,were cultured in the presence of 10ng/ml recombinant murine GM-CSF and 1ng/ml murine IL-4.The nonadherent cells were gently washed off after 3 day,s culture,the remained clusters were cultured foe 4 days;on day 8,CD11c+ mature DC were positively selected using CD11c magnetic microbeads.These mature DCs express high levels of MHC-II,CD80,CD86,CD40 molecules and had potent ability of stimulating auto and allogenic T cell proliferation. LPS promoted DCs to be mature and secrete higher level of IL-12(p70)、IL-6、TNF-α和IL-1β.3.The matured DCs were seeded on the monolayer when the stromal cells teached 50% confluence,and can be reversedly turned to proliferative progenitors in the presence of DSC.【Conclution】1. Abortive decidual tissues were digested with trypsin,hyalace and collagenase.All components of the decidual cells were cultured together.The present method is simple,economical and practical.As a result,high purity DSC can be obtained with the isolation procedures. The construction of a co-coltured system of decidual cells will lay a good foundation to study pathological pregnancy.2. A large amount of mature DCs could be generated by in vitro culture of mice bone marrow cells. Biological Characteristic of DC was indentificated by morphological observation , Relatively Specific surface phenotype and potent ability of stimulating auto and allogenic T cell proliferation.Then it will lay a foundation for Related Fundamental Research and clinical application of DC.3.The mature DC can be reversedly turned to proliferative progenitors in the presence of DSC,and DSC can protect DCs from activation-induted apoptosis or cell death. The results show that stromal microenvironment composition of DSC effected natural outcome of mature DCs and which indicated that regulation of immune cells by Maternal-Fetal Interface immune microenvironment played an important role in inducing Maternal-Fetal immune tolerance.更多还原