小鼠骨髓树突状细胞对白念珠菌吞噬、杀伤作用的研究

摘要/Abstract

摘要：

目的体外分离培养小鼠骨髓树突状细胞（Dendritic Cell，DC）并与白念珠菌共同孵育，研究不同浓度的DC对白念珠菌的吞噬、杀伤作用，并研究随着共孵育时间的延长DC表型的变化及其吞噬、杀伤白念珠菌能力的变化。方法无菌条件下分离培养C57小鼠骨髓DC；倒置显微镜下观察DC的形态；Cellometer Mini细胞计数仪测定DC生长曲线及细胞直径变化；流式细胞仪检测DC及白念珠菌刺激后DC表面标志物CD11c及其表面分子MHCⅡ、CD40、CD80及CD86的表达；光学显微镜、激光共聚焦显微镜及流式细胞仪测定DC对白念珠菌的吞噬及杀伤作用。结果体外培养10 d小鼠骨髓DC，倒置显微镜观察可见典型DC形态；随着培养时间的延长，细胞逐渐增大，培养第10天时，细胞直径分布最多的为10.2 μm；生长曲线显示，DC呈倍数增长，第2～9天为细胞对数生长期，第9天左右增殖曲线达到顶峰。培养第10天，未经白念珠菌刺激的DC表面CD11c分子及CD80分子高水平表达，CD40、CD86及MHCⅡ低表达；经白念珠菌刺激后，DC表面CD11c分子、CD80、CD40、CD86及MHCⅡ均高表达，呈成熟DC表型。激光共聚焦（Laser Scanning Confocal Microscope，LSCM）及流式细胞仪（Flow Cytometer，FCM）检测DC对白念珠菌吞噬作用，当DC与白念珠菌的比例为1：1时，随着共培养时间的延长（30 min～1.5 h），吞噬率无明显变化（P=0.063）；当DC与白念珠菌比例为2：1、1：2及1：4，随着DC细胞所占比例的减小，DC细胞吞噬作用增强，当比例为1：4时，DC吞噬作用最强，且随着共培养时间的延长（30 min～1.5 h），吞噬率也明显上升，差异有统计学意义（P=0.003；P=0.002；P=0.002）。显微镜检测DC对白念珠菌杀伤率，可见随着时间的延长，各实验组的杀伤率下降（P=0.000）；当DC：白念珠菌=1：1时，较2：1时，杀伤率明显下降，但随着白念珠菌比例在一定范围内的增高（从1：1到1：4），杀伤率明显增高，差异有统计学意义（P=0.000）。结论小鼠骨髓细胞体外经GM-CSF诱导可培养DC具有较高的纯度，呈未成熟细胞表型；且每只C57小鼠可获得3×10⁷总数的细胞，可满足一些实验的DC用量。经白念珠菌刺激后DC表型成熟；在一定的比率范围内，DC对白念珠菌有较强的吞噬及杀伤作用，其吞噬作用随着DC与白念珠菌比例的减小而增强。且随着两者作用时间的延长，DC对白念珠菌的吞噬作用逐渐增强而杀伤作用逐渐减弱。

关键词: 骨髓, 树突状细胞, 白念珠菌, 吞噬, 杀伤

Abstract:

Objective Mouse bone marrow-derived dendritic cells were isolated and cultured in vitro and co-incubated with Candida albicans,in order to study the phenotype,phagocytosis and cytotoxicity of which against Candida albicans under different concentration and time. Methods Dendritic cells from C57 mouse bone marrow were isolated and cultured under aseptic conditions.The morphological and the growth curve and cell diameter of DC were observed by optical microscope and Cellometer Mini cell counter.The expression levels of CD11c,MHCII,CD40,CD80 and CD86 of DC were measured by flow cytometer.The phagocytosis and cytotoxicity of DC against Candida albicans were detected by microscope,Laser Scanning Confocal Microscope(LSCM) and flow cytometry. Results Typical morphology of DC cells was observed under inverted microscope 10 days after culture.10.2 μm was the most widely distributed diameter of DC as illustrated and the size of DC increased with the extension of incubation time.The growth curve showed that DC increased multiply.After inoculation,2-9 days were logarithmic phase and stagnate phase was from 9 days.The result of immunological phenotype detected by FCM showed that the CD11c and CD80 were highly expressed and MHCII,CD40,CD86 were lower expressed in control group.After stimulated by Candida albicans,the DC were appeared mature phenotype and CD11c,CD80,CD40,CD86 and MHCⅡ were all highly expressed.As the ratio(DC:Candida albicans) reduced(from 2:1 to 1:4),the phagocytosis effects of DC enhanced and the strongest effect appeared when the ratio was 1:4.When the ratio was 1:1,the phagocytosis effects of DC was no significant change(P=0.063) with the extension of co-incubation time(30 min to 1.5 h).But when the ratio were 2:1,1:2 and 1:4,the phagocytosis effects of DC were obviously rised with the extension of co-incubation time(30 min to 1.5 h),a statistically significant difference(P=0.003,P=0.002,P=0.002).When co-incubation time was prolonged,the cytotoxicity of DC was dramatic descend(P=0.000) detected by microscope.When the ratio(DC:Candida albicans) was 1:1,compared with the 2:1 group,the cytotoxicity effects of DC was dramatic declined.But when the ratio of Candida albicans increased within limits(from 1:1 to 1:4),the cytotoxicity was significantly increased(P=0.000),with statistical significance.Conclusion Dendritic cells isolated from mouse bone marrow had high purity and immature cell phenotype.We could obtain 3×10⁷ cells per C57 mouse DC which could meet the requirements of experiments very well.After stimulated by Candida albicans,mature DCs were obvious increased.DC had strong phagocytosis and cytotoxicity against Candida albicans,and the effects of phagocytosis and cytotoxicity enhanced with the ratio(DC:Candida albicans) reduced.When co-incubation time was prolonged,the phagocytosis of DC against Candida albicans was increased and the cytotoxicity was decreased.

Key words: bone marrow, dendritic cells, Candida albicans, phagocytosis, cytotoxicity

中图分类号:

R379.4

王琼, 史冬梅, 刘维达. 小鼠骨髓树突状细胞对白念珠菌吞噬、杀伤作用的研究[J]. 中国真菌学杂志, 2016, 11(4): 193-200.

WANG Qiong, SHI Dong-mei, LIU Wei-da. The study of mouse bone marrow-derived dendritic cells phagocytosis and cytotoxicity against Candida albicans in vitro[J]. Chinese Journal of Mycology, 2016, 11(4): 193-200.

参考文献

[1] Zeng Z,liu X,Jiang Y,et al.Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells[J].Cell Biochem Biophys,2006,45(1):19-30.
[2] Perruccio K,Boza S,Montagnoli C,et al.Prospects for dendritic cell vaccination against fungal infections in hematopoetic transplantation[J].Blood Cells,2004,33(3):248-255.
[3] Gafa V,Remoli MR,Giacomini E,et al.In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment[J].Microb Infect,2007,9(8):971-980.
[4] Shi D,Li D,Yin Q,et al.Silenced suppressor of cytokine signaling 1(SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro[J].Immunol Res,2015,61(3):206-218.
[5] She X,Zhang L,Chen H,Calderone R,Li D.Cell surface changes in the Candida albicans mitochondrial mutant goa1Delta are associated with reduced recognition by innate immune cells[J].Cell Microbiol,2013,15(9):1572-1584.
[6] Steinman RM,Idoyaga J.Features of the dendritic cell linesge[J].Immunol Rev,2010,234(1):5-17.
[7] Li Y,Zeng X,He L,et al.Dendritic cell activation and maturation induced by recombinant calreticulin fragment 39-272[J].Int J Clin Exp Med,2015,8(5):7288-7296.
[8] Magalhães YC,Bomfim MR,Melônio LC,et al.Clinical significance of the isolation of Candida species from hospitalized patients[J].Braz J Microbiol,2015,46(1):117-123.
[9] Wang Y,Liang Y,Zhang Y,et al.Bortezomib inhibits bone marrow-derived dendritic cells[J].Int J Clin Exp Pathol,2015,8(5):4857-4862.
[10] Steinman RM,Chon ZA.Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology,quantitation,tissue distribution[J].J Exp Med,1973,137(5):1142-1162.
[11] Fidan I,Kalkanci A,Yesilyurt E,et al.In vitro effects of Candida albicans and Aspergillus fumigatus on dendritic cells and the role of beta glucan in this effect[J].Adv Clin Exp Med,2014,23(1):17-24.
[12] Reschner A,Hubert P,Delvenne P,et al.Innate lymphocyte and dendritic cell cross-talk:a key factor in the regulation of the immune response[J].Clin Exp Immunol,2008,152(2):219-226.

[1]	刘丹, 邢丽枝, 杨心茹, 罗伟, 何荣霞. 唑类药物治疗外阴阴道假丝酵母菌病研究进展[J]. 中国真菌学杂志, 2020, 15(5): 318-320.
[2]	马玉帛, 张逸, 黄晨, 王晓雯, 李若瑜. 自然杀伤细胞在真菌感染免疫的研究进展[J]. 中国真菌学杂志, 2020, 15(3): 175-178.
[3]	邱熙然, 陈思敏, 侯炜彤, 张玉, 郭诗雨, 姜远英, 安毛毛. 白念珠菌侵袭宿主毒力因子研究进展[J]. 中国真菌学杂志, 2020, 15(3): 183-188.
[4]	张静云, 彭靖雯, 王琼, 高盈, 陈万鑫, 沈永年, Li Dong-mei, 佘晓东, 刘维达. 表皮生长因子受体在阴道念珠菌病发病过程中的作用初探[J]. 中国真菌学杂志, 2020, 15(2): 65-71.
[5]	刘光荣, 翟春涛, 金敏蓉, 李成亮. 天然抗菌剂对5种致病菌的抑制作用研究[J]. 中国真菌学杂志, 2020, 15(2): 88-92.
[6]	谭静文, 徐红, 宋金凤, 高志琴, 杨虹, 杨连娟, 温海. 聚六亚甲基双胍体外抗真菌作用研究[J]. 中国真菌学杂志, 2020, 15(2): 93-96.
[7]	张静云, 佘晓东, 刘维达. 白念珠菌糖代谢与毒力[J]. 中国真菌学杂志, 2020, 15(1): 61-64.
[8]	王晓东, 迪丽努尔·塔西买买提, 迪丽达尔·地里夏提, 哈地利亚·哈斯木, 帕丽达·阿布利孜. 球形孢子丝菌刺激小鼠树突状细胞表达前炎症细胞因子的研究[J]. 中国真菌学杂志, 2019, 14(6): 321-325.
[9]	赵晓琴, 冯文莉. 转录因子在白念珠菌生物膜形成过程中的调控机制[J]. 中国真菌学杂志, 2019, 14(6): 381-384.
[10]	王钰婷, 王玉柱, 刘锦燕, 孟玲宁, 李卫华, 项明洁. 酵母相和菌丝相白念珠菌感染阴道上皮细胞免疫反应的比较[J]. 中国真菌学杂志, 2019, 14(4): 193-199.
[11]	蔡晴, 杨连娟, 高志琴, 任弘瑾, 杨虹, 谭静文. 罗米地辛对氟康唑体外抗白念珠菌影响的研究[J]. 中国真菌学杂志, 2019, 14(4): 200-203,216.
[12]	葛力瑜, 梁官钊, 刘维达. 抗黏膜白念珠菌感染的固有免疫研究进展[J]. 中国真菌学杂志, 2019, 14(4): 249-252.
[13]	姬名硕, 王晓东, 帕丽达·阿布利孜. NLRP3炎症小体在真菌感染中的作用机制研究进展[J]. 中国真菌学杂志, 2019, 14(4): 253-256.
[14]	施高翔, 姜晶晶, 汪云霞, 赵婷, 邵菁, 汪天明, 汪长中. 苦参-蛇床子药对提取物对白念珠菌VVC临床株的抑制作用研究[J]. 中国真菌学杂志, 2019, 14(3): 147-153.
[15]	沈煜宸, 曹毅, 陶茂灿. 白念珠菌生物膜耐药机制及中药治疗的研究进展[J]. 中国真菌学杂志, 2019, 14(3): 176-179.