CAJ | 학술논문

目的以小鼠骨髓细胞为前体,建立一种高效、简便的体外扩增、分离培养树突状细胞(DC)的方法 .方法实验分为GM/4组和GM/4-α组,以重组鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白介素-4(rmIL-4)对小鼠骨髓细胞联合诱导培养,GM/4-α组在第5天添加rmTNF-α继续培养48 h,GM/4组不加并于第5天时终止培养;分别收集第5天、第7天的悬浮及疏松贴壁细胞,扫描电镜观察细胞形态,流式细胞仪测定细胞表面分子,同种异体混合淋巴细胞反应(MLR),检测2组细胞刺激同种异体T细胞增殖的能力.结果所收集的2组细胞均具有典型DC形态,细胞表面高表达小鼠髓源性DC相对特异性标志CD11c,表达率达75%以上;GM/4组DC细胞表面CD40、CD86、MHC-Ⅱ的表达率分别为30.5%、34.2%、45.1%,GM/4-α组则分别为78.7%、88.3%、96.7%;MLR中GM/4组DC刺激同种异体T细胞活化增殖的能力不如OM/4-α组强.结论此种方法能于体外定向诱导和扩增出大量髓源性DC,这为后续研究DC在器官移植后诱导机体免疫耐受的机制奠定了基础.
목적 이소서골수세포위전체,건립일충고효、간편적체외확증、분리배양수돌상세포(DC)적방법 .방법 실험분위GM/4조화GM/4-α조,이중조서립세포-거서세포집락자격인자(rmGM-CSF)화중조서백개소-4(rmIL-4)대소서골수세포연합유도배양,GM/4-α조재제5천첨가rmTNF-α계속배양48 h,GM/4조불가병우제5천시종지배양;분별수집제5천、제7천적현부급소송첩벽세포,소묘전경관찰세포형태,류식세포의측정세포표면분자,동충이체혼합림파세포반응(MLR),검측2조세포자격동충이체T세포증식적능력.결과 소수집적2조세포균구유전형DC형태,세포표면고표체소서수원성DC상대특이성표지CD11c,표체솔체75%이상;GM/4조DC세포표면CD40、CD86、MHC-Ⅱ적표체솔분별위30.5%、34.2%、45.1%,GM/4-α조칙분별위78.7%、88.3%、96.7%;MLR중GM/4조DC자격동충이체T세포활화증식적능력불여OM/4-α조강.결론 차충방법 능우체외정향유도화확증출대량수원성DC,저위후속연구DC재기관이식후유도궤체면역내수적궤제전정료기출.
Objective To establish an effective and convenient method in vitro for induction and amplification of dendritic cell (DC)by using mouse bone marrow cell as the precursor cell.Methods The experiment were divided into two groups:GM/4 and GM/4-α.The precursor cell were cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor(rmGM-CSF)and interleukin-4(rmIL-4)in vitro,and dendritic cell in the GM/4-α group were treated with recombinant mouse tumor necrosis factor-alpha(rmTNF-α)on the fifth day for stimulating forty-eight hours and that in the GM/4 group was used as controls without treatment.The suspending and loosely adherent cell were collected on the fifth and seventh days for examing with scanning electronic microscope and flow cytometry,and their capacity to stimulate allogenetic T cell proliferation was observed by mixed lymphocyte reaction(MLR).Results The two group cell exhibited typical morphological characteristics of DC and had a much higher CD11c exDression rate,which was over 75%.Otherwise,GM/4 DC had a lower expression rate of CD40、CD86、MHC-Ⅱ,which was 30.5%、34.2%、45.1% and that in GM/4-α DC was 78.7%、88.3%、96.7% respectively.In MLR,GM/4 DC had the weaker ability for stimulating the proliferation of allogenetic T cell compared with GM/4-α DC.Conclusion The DC could be induced and amplified from mouse bone marrow by using the method,which laid foundation for the further study on the mechanism of DC inducing immunological tolerance after organ transplantation.