Lewis大鼠骨髓来源成熟和未成熟树突状细胞的提取与鉴别  

Extraction and differentiation of mature and immature dendritic cells from Lewis rat bone marrow

作　　者：李立强 李明皓[2] 李良[1] 李文波[1] 张军[1] 孔令梅 Li Liqiang;Li Minghao;Li Liang;Li Wenbo;Zhang Jun;Kong Lingmei(Department of Hepatobiliary Surgery,Second People’s Hospital of Hefei/Department of Hepatobiliary Surgery,Hefei Hospital Affiliated to Anhui Medical University/Teaching Hospital of Second People’s Hospital of Hefei Affiliated to Bengbu Medical College,Hefei 230011,Anhui Province,China;Department of Hepatobiliary Surgery,People’s Hospital of Ningxia Hui Autonomous Region,Yinchuan 750002,Ningxia Hui Autonomous Region,China)

机构地区：[1]合肥市第二人民医院肝胆外科/安徽医科大学附属合肥医院肝胆外科/蚌埠医学院附属合肥市第二人民医院教学医院,安徽省合肥市230011 [2]宁夏回族自治区人民医院肝胆外科,宁夏回族自治区银川市750002

出　　处：《中国组织工程研究》2024年第25期4013-4017,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金地区基金(81560114),项目负责人:李明皓;宁夏自然科学基金(2021AAC03308),项目负责人:李明皓;蚌埠医学院科技项目自然科学重点项目(2022byzd200),项目负责人:张军;蚌埠医学院科技项目自然科学重点项目(2022byzd199),项目负责人:李良。

摘　　要：背景:树突状细胞在未成熟阶段表现出极强的抗原吞噬功能,它可以在免疫耐受、癌症的免疫治疗等方面都表现出极大的优越性。但由于未成熟树突状细胞在生物体内含量极微,这就严重限制了它在临床、科研方面的应用。目的:提取鉴别Lewis大鼠骨髓来源成熟和未成熟树突状细胞。方法:从Lewis大鼠骨髓中分离骨髓前体细胞,应用20 ng/mL粒细胞集落刺激因子、10 ng/mL白细胞介素4培养7 d诱导为未成熟树突状细胞,然后在未成熟树突状细胞中加入1μg/mL脂多糖继续培养2 d诱导为成熟树突状细胞。采用荧光倒置显微镜观察树突状细胞形态,流式细胞仪鉴定成熟和未成熟树突状细胞表面特异性分子,ELISA检测成熟和未成熟树突状细胞培养上清白细胞介素10、白细胞介素12和白细胞介素17A因子的分泌水平,混合淋巴细胞反应检测成熟和未成熟树突状细胞对T淋巴细胞的刺激反应。结果与结论:①普通荧光倒置显微镜下观察树突状细胞具有明显的突起结构;②流式细胞仪可见未成熟树突状细胞低表达CD40、CD86等共刺激分子;相反,成熟树突状细胞高表达上述共刺激分子;③未成熟树突状细胞的白细胞介素10、白细胞介素17A分泌水平要远高于成熟树突状细胞(P<0.01);未成熟树突状细胞的白细胞介素12分泌水平低于成熟树突状细胞(P<0.05);④成熟树突状细胞对T细胞的刺激明显比成熟树突状细胞强,当成熟树突状细胞与T淋巴细胞比例达到1∶10时刺激能力更强;⑤结果表明:Lewis大鼠骨髓前体细胞可以分化为树突状细胞,通过流式细胞鉴定、相关因子检测、混合淋巴细胞反应能够鉴别树突状细胞的成熟和未成熟状态。BACKGROUND:Dendritic cells exhibit extremely strong antigen phagocytic function in the immature stage,and they can demonstrate great advantages in immune tolerance,cancer immunotherapy,and other aspects.However,due to the extremely low content of immature dendritic cells in living organisms,its clinical and scientific applications are severely limited.OBJECTIVE:To study the extraction and identification of mature and immature dendritic cells from Lewis rat bone marrow.METHODS:Bone marrow precursor cells were isolated from the bone marrow of Lewis rats,and immature dendritic cells were induced by 20 ng/mL of granulocyte colony-stimulating factor and 10 ng/mL of interleukin-4 for 7 days,and then mature dendritic cells were induced by adding 1μg/mL of lipopolysaccharide to immature dendritic cells for 2 days.The morphology of dendritic cells was observed using inverted fluorescence microscopy.The surface-specific molecules of mature and immature dendritic cells were identified by flow cytometry,and the secretion levels of supernatant interleukin-10,interleukin-12,and interleukin-17A in mature and immature dendritic cells were detected by ELISA.The response of mature and immature dendritic cells to T lymphocyte stimulation was measured by mixed lymphocyte reaction.RESULTS AND CONCLUSION:(1)The dendritic cells showed an obvious protrusion structure under an ordinary inverted fluorescence microscope.(2)Flow cytometry showed low expression of CD40,CD86,and other co-stimulatory molecules in immature dendritic cells.On the contrary,mature dendritic cells highly expressed the above co-stimulatory molecules.(3)The secretion of interleukin-10 and interleukin-17A in immature dendritic cells was much higher than that in mature dendritic cells(P<0.01).Interleukin-12 secretion in immature dendritic cells was much lower than that in mature dendritic cells(P<0.05).(4)Mature dendritic cells stimulated T cells significantly better than immature dendritic cells,and the stimulation ability was stronger when the ratio of mature dendri

关 键 词：树突状细胞 未成熟树突状细胞 成熟树突状细胞 细胞因子 混合淋巴细胞反应 

分 类 号：R459.9[医药卫生—治疗学] R392-3[医药卫生—临床医学] R617