细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究

Study on the polarization of MDSC stimulated by Echinococcus granulosus protoscolex-derived exosomes in vitro

目的探究细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞(MDSC)向M2型巨噬细胞极化的动态变化,并探讨二者协同发挥免疫抑制作用的生物学意义。方法体外培养细粒棘球蚴原头节并收集培养上清,超速离心上清获得外泌体。透射电镜观察外泌体形态,蛋白质免疫印迹(Western blotting)检测外泌体蛋白CD9和烯醇化酶的表达。取3只健康BALB/c小鼠,制备股骨髓系细胞,刺激分化为MDSC后,分为实验组、阳性对照组和阴性对照组。于6孔板中每孔加入1×10^(6)个MDSC细胞,实验组、阳性对照组和阴性对照组分别加入20μl外泌体(5μg)、白细胞介素-4(IL-4)(40 ng)和RPMI 1640培养基进行刺激。于刺激24、48和72 h后,分别收集3组MDSC,采用流式细胞术分析单核型MDSC(M-MDSC)的细胞比例变化及其M2型巨噬细胞分子标志F4/80和CD206的表达情况。采用GraphPad Prism 8.0.2统计学软件进行统计学分析。结果透射电镜下可见细粒棘球蚴原头节源外泌体为圆形的膜状结构,直径集中在60~90 nm。Western blotting分析结果显示,外泌体有特异性蛋白CD9和烯醇化酶的表达。外泌体刺激MDSC 24 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为(77.14±0.78)%、(75.74±0.50)%,与阴性对照组的(78.13±0.81)%比较差异均无统计学意义(t=1.24、3.45,P>0.05);刺激48 h后,阴性对照组、实验组和阳性对照组M-MDSC占MDSC的比例分别为(80.47±0.85)%、(78.27±0.69)%和(68.01±1.33)%,其中实验组与阴性对照组比较差异无统计学意义(t=2.48,P>0.05),阳性对照组与阴性对照组比较差异有统计学差异意义(t=11.20,P<0.01);刺激72 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为(50.03±0.59)%和(46.14±0.87)%,均低于阴性对照组的(74.94±1.53)%,差异有统计学意义(t=21.52、23.18,P<0.01)。外泌体刺激MDSC 24 h、48 h和72 h后,实验组和阳性对照组M-MDSC上表达M2型巨噬细胞分子标志的比例分别为(11.83±0.06)%、(12.48±0.12)%,(15.11±0.21)%、(15.42±0.27)%和(29.12±1.34)%、(31.83±1.27)%,均高于阴性对照组的(8.52±1.11)%、(8.79±0.35)%和(15.49±0.26)%,差异均有统计学意义(P<0.05或P<0.01)。结论细粒棘球蚴原头节源外泌体体外可调控M-MDSC向M2型巨噬细胞的极化,且极化随刺激时间进程不断推进。

Objective To investigate the dynamic changes of the polarization of myeloid-derived suppressor cells(MDSC)to M2 macrophages stimulated by Echinococcus granulosus protoscolex-derived exosomes,and to explore the biological significance of their synergistic immunosuppressive effect.Methods The exosomes were collected by ultracentrifugation of supernatant of E.granulosus protoscoleces in vitro culture.The morphology of exosomes was observed by transmission electron microscope,and the expression of exosomal protein CD9 and enolase were detected by Western blotting.Subsequently,MDSC stimulated from myeloid cells of 3 healthy BALB/c mice femoral bone marrow were added at 1×10^(6)MDSC per well of a 6-well plate,and divided into experimental group,positive control group and negative control group.20μl exosomes(5μg),20μl IL-4(40 ng)and 20μl RPMI 1640 medium were added respectively for co-culture.The proportions of monocytic DMSC(M-MDSC)and the expression of M2macrophages molecular markers F480 and CD206 in co-cultured cells at 24 h,48 h,and 72 h,were analyzed by Flow cytometry,respectively.Statistical analysis was performed by statistical software GraphPad Prism 8.0.2.Results E.granulosus protoscolex-derived exosomes are of circular membrane structure with a diameter of 60-90 nm by transmission electron microscopy.The expression of specific protein CD9 and enolase in exosomes was detected by Western blotting.The proportions of M-MDSC in MDSC in the negative control,experimental and positive control group were(78.13±0.81)%,(77.14±0.78)%,and(75.74±0.50)%,respectively after 24 h of stimulation by exosomes.The differences were not significant(P>0.05).The proportions of M-MDSC in the negative control,experimental and positive control group were(80.47±0.85)%,(78.27±0.69)%,and(68.01±1.33)%,respectively,after48 h of stimulation,showed no significant difference between experimental group and negative control group(P>0.05),however significant difference was observed between the positive control and the negative control group(P<0.01).After 72 h,the proportions of M-MDSC in the experimental and positive control groups in MDSC were(50.03±0.59)%and(46.14±0.87)%,which are lower than negative control group(74.94±1.53)%,and the differences were statistically significant(P<0.001).After stimulation with exosomes for 24 h,48 h,and 72 h,the proportions of M-MDSC expressing M2 macrophages molecular markers in experimental and positive control group were(11.83±0.06)%,(12.48±0.12)%;(15.11±0.21)%,(15.42±0.27)%;and(29.12±1.34)%,(31.83±1.27)%,respectively,which were higher than those in the negative control group(8.52±1.11)%,(8.79±0.35)%,and(15.49±0.26)%,and all differences were statistically significant(P<0.05 or P<0.01).Conclusion E.granulosus protoscolex-derived exosomes could regulate the polarization of M-MDSC to M2 macrophages,and the polarization continued to advance with the stimulation time.