多房棘球蚴源性外泌体对巨噬细胞极化的影响

冶赓博; 陈功富; 崔紫烟; 吴俊杰; 黄登亮; 尹凤娇; 王志鑫; 于文昊; 孔繁玉; 樊海宁; 任利

doi:10.3969/j.issn.1001-5256.2023.04.019

多房棘球蚴源性外泌体对巨噬细胞极化的影响

DOI: 10.3969/j.issn.1001-5256.2023.04.019

冶赓博^{1, 2},
陈功富^{1, 2},
崔紫烟^{1, 2},
吴俊杰^{1, 2},
黄登亮²,
尹凤娇^{1, 2},
王志鑫^{1, 2},
于文昊^{1, 2},
孔繁玉^{1, 2},
樊海宁^{1, 2},
任利^{1, 2, , ,}

1.
青海大学附属医院肝胆胰外科，西宁 810001
2.
青海省包虫病研究重点实验室，西宁 810001

基金项目:

青海省科技厅项目 (2019-ZJ-7031)

伦理学声明：本研究于2019年7月3日经青海大学附属医院科研伦理委员会审批，批号为P-SL-2019042, 符合实验室动物管理与使用准则。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：冶赓博、陈功富负责课题设计，撰写论文；黄登亮、崔紫烟、吴俊杰负责实验实施，数据整理；孔繁玉、于文昊、尹凤娇查阅文献，分析数据；任利、樊海宁、王志鑫负责课题设计，指导撰写论文并最后定稿。

详细信息

通信作者:
任利, renliweimin_xn@126.com (ORCID: 0000-0001-6306-3533)

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出版历程
- 收稿日期: 2022-09-27
- 录用日期: 2022-11-22
- 出版日期: 2023-04-20

Effect of exosomes derived from Echinococcus multilocularis on macrophage polarization: A preliminary study

Gengbo YE^{1, 2},
Gongfu CHEN^{1, 2},
Ziyan CUI^{1, 2},
Junjie WU^{1, 2},
Dengliang HUANG²,
Fengjiao YIN^{1, 2},
Zhixin WANG^{1, 2},
Wenhao YU^{1, 2},
Fanyu KONG^{1, 2},
Haining FAN^{1, 2},
Li REN^{1, 2
, ,
,}

1.
Department of Hepatopancreatobiliary Surgery, The Affiliated Hospital of Qinghai University, Xining 810001, China
2.
Qinghai Province Key Laboratory of Hydatid Disease Research, Xining 810001, China

Research funding:

Project of Qinghai Provincial Department of Science and Technology (2019-ZJ-7031)

More Information

Corresponding author: REN Li, renliweimin_xn@126.com (ORCID: 0000-0001-6306-3533)

摘要

摘要: 目的探究不同时间及浓度多房棘球蚴源性外泌体对巨噬细胞极化的影响。方法从60只造模BALB/c小鼠中随机选取4只，应用7.0T MRI观察腹腔病灶生长情况；解剖造模小鼠，通过腹腔病灶提取原头节进行体外培养，超速离心法从培养上清中提取外泌体，透射电镜及蛋白质免疫印迹法鉴定外泌体表征。将未加外泌体处理的巨噬细胞单独培养组设为对照组，不同浓度多房棘球蚴来源外泌体与巨噬细胞共培养组设为实验组(10 μg/mL组、50 μg/mL组)，分别共培养48 h和72 h，显微镜下观察巨噬细胞形态变化。通过流式细胞术和酶联免疫吸附实验(ELISA)检测极化状态。符合正态分布的计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果 7.0T MRI显示小鼠腹腔内弥漫分布、大小不等的病灶形成；多房棘球蚴源性外泌体直径100 nm左右，呈杯型或茶托型，其表面标志物CD9、TSG101和CD63表达阳性。共培养后，实验组大部分细胞拉长，形态不规则，主要呈多角形；流式细胞术检测发现，共培养48 h，对照组CD16/32、CD206、CD369阳性率分别为(99.53±0.06)%、(90.27±0.21)%、(2.40±0.20)%；与对照组相比，除10 μg/mL外泌体组CD369阳性率[(0.80±0.00)%]降低(P＜0.05)，其余组别CD16/32、CD206、CD369阳性率均明显升高(P值均＜0.000 1)；共培养72 h，对照组CD16/32、CD206、CD369阳性率分别为(99.67±0.06)%、(85.47±0.55)%、(6.60±0.20)%，实验组CD16/32、CD206、CD369阳性率较对照组均明显升高(P值均＜0.05)。ELISA结果示：共培养48 h，对照组IL-6及TNFα水平分别为(58.53±15.52) pg/mL、(320.70±5.30)pg/mL，实验组外泌体浓度为50 μg/mL时IL-6[(98.81±15.55) pg/mL]较对照组升高(P＜0.05)；共培养72 h，对照组IL-6及TNFα水平分别为(76.22±9.68)pg/mL、(323.90±87.37)pg/mL，当外泌体浓度为10 μg/mL时TNFα水平[(164.20±14.17)pg/mL]较对照组明显下降(P＜0.05)；当外泌体浓度为50 μg/mL时IL-6水平[(99.52±8.35)pg/mL]较对照组升高(P＜0.05)。结论多房棘球蚴源性外泌体可调控巨噬细胞极化，且在浓度为10 μg/mL，共培养72 h后，可导致巨噬细胞M2样极化，具体方式有待进一步研究。
- 棘球蚴病, 肝 /
- 外泌体 /
- 巨噬细胞 /
- 小鼠，近交BALB C
Abstract: Objective To investigate the effect of exosomes derived from Echinococcus multilocularis on macrophage polarization after treatment for different durations and concentrations. Methods A total of 60 BALB/c mice were used for modeling, among which 4 mice were selected to observe the growth of abdominal lesions on 7.0T MRI. The mice for modeling were dissected, and the protoscoleces was taken from the abdominal lesion and cultured in vitro; ultracentrifugation was used to extract the exosomes from the supernatant, and transmission electron microscopy and Western blotting were used for the characterization of exosomes. The macrophages without exosome treatment were established as control group, and the macrophages co-cultured with different concentrations of exosomes derived from Echinococcus multilocularis were established as experimental group (10 μg/mL group and 50 μg/mL group) and were cultured for 48 and 72 hours. The morphological changes of macrophages were observed under a microscope, and flow cytometry and ELISA were used to observe polarization state. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results The results of 7.0T MRI showed the formation of diffuse lesions with different sizes in the abdominal cavity of mice, and the exosomes derived from Echinococcus multilocularis were approximately 100 nm in diameter and were cup-shaped or saucer-shaped, with the positive expression of the surface markers CD9, TSG101, and CD63. After co-culture, most of the cells in the experimental group were elongated with an irregular and polygonal shape. Flow cytometry showed that after 48 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.53%±0.06%, 90.27%±0.21%, and 2.40%±0.20%, respectively; compared with the control group, except that the 10 μg/mL exosome group had a significant reduction in the positive rate of CD369 (0.80%±0.00%) (P < 0.05), all the other groups had a significant increase in the positive rates of CD16/32, CD206, and CD369 (all P < 0.000 1); after 72 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.67%±0.06%, 85.47%±0.55%, and 6.60%±0.20%, respectively, and compared with the control group, the experimental group had significant increases in the positive rates of CD16/32, CD206, and CD369 (all P < 0.05). ELISA showed that after 48 hours of co-culture, the levels of IL-6 and TNFα in the control group were 58.53±15.52 pg/mL and 320.70±5.30 pg/mL, respectively, and when the exosome concentration was 50 μg/mL, the level of IL-6 in the experimental group was 98.81±15.55 pg/mL, which was higher than that in the control group (P < 0.05); after 72 hours of co-culture, the levels of IL-6 and TNFα in the control group were 76.22±9.68 pg/mL and 323.90±87.37 pg/mL, respectively, and when the exosome concentration was 10 μg/mL, the level of TNFα was 164.20±14.17 pg/mL, which was significantly lower than that in the control group (P < 0.05); when the exosome concentration was 50 μg/mL, the level of IL-6 was 99.52±8.35 pg/mL, which was significantly higher than that in the control group (P < 0.05). Conclusion Exosomes derived from Echinococcus multilocularis can regulate macrophage polarization and induce M2-like polarization of macrophages after co-culture at a concentration of 10 μg /mL for 72 hours, and further studies are needed to clarify the specific method.
- Echinococcosis, Hepatic /
- Exosomes /
- Macrophages /
- Mice, Inbred BALB C

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图 1 7.0T MRI在模型小鼠中的应用

注：a, T₁WI像；b, T₂WI像。

Figure 1. Application of 7.0T MRI in mouse model

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图 2 多房棘球蚴源性外泌体的鉴定

注：a, TEM观察多房棘球蚴形态；b, Western Blot检测相关标志物表达。

Figure 2. Identification of exosomes derived from echinococcus multilocularis

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图 3 不同浓度外泌体与J774A.1细胞共培养的形态变化(×200)

Figure 3. Morphological changes of J774A.1 cells co-cultured with different concentrations of exosomes (×200)

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图 4 流式细胞术检测不同时间及浓度外泌体刺激巨噬细胞CD16/32、CD206、CD369表达量

Figure 4. The expressions of CD16/32, CD206 and CD369 in macrophages stimulated by exosomes at different time and concentration were detected by flow cytometry

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图 5 共培养后CD16/32、CD206、CD369变化趋势

注：与对照组比较，*＜0.05, ****P＜0.000 1。

Figure 5. The change trend of CD16/32, CD206 and CD369 after co-culture

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图 6 ELISA检测不同浓度的外泌体刺激巨噬细胞后相关分子表达情况

注：a, 48 h; b, 72 h。与对照组比较，*P＜0.05。

Figure 6. ELISA was used to detect the expression of related molecules in macrophages stimulated by exosomes at different concentrations

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图 7 M2/M1型巨噬细胞相关分子比值变化趋势

注：与对照组比较，***P＜0.001，****P＜0.000 1。

Figure 7. The trends in the molecular ratio of M2/M1 macrophages

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表 1 外泌体与巨噬细胞共培养48 h流式结果

Table 1. Flow cytometry results of exosomes co-cultured with macrophages for 48 hours

分组	CD16/32(%)		CD206(%)		CD369(%)
分组	48 h	72 h	48 h	72 h	48 h	72 h
对照组	99.53±0.06	99.67±0.06	90.27±0.21	85.47±0.55	2.40±0.20	6.60±0.20
10 μg/mL组	100.00±0.00²⁾	99.83±0.06¹⁾	98.67±0.21²⁾	96.77±0.15²⁾	0.80±0.00¹⁾	16.67±0.45²⁾
50 μg/mL组	100.00±0.00²⁾	99.83±0.06¹⁾	98.70±0.20²⁾	96.03±0.23²⁾	4.50±0.30²⁾	9.03±0.23¹⁾
F值	196.00	8.33	1 678.00	946.90	238.40	836.90
P值	＜0.000 1	＜0.05	＜0.000 1	＜0.000 1	＜0.000 1	＜0.000 1
注：与对照组比较，1)P＜0.05, 2)P＜0.000 1。

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表 2 外泌体与巨噬细胞共培养ELISA结果

Table 2. ELISA results of exosomes co-cultured with macrophages

组别	IL-6(pg/mL)		TNFα(pg/mL)
组别	48 h	72 h	48 h	72 h
对照组	58.53±15.52	76.22±9.68	320.70±5.30	323.90±87.37
10 μg/mL组	52.19±13.16	76.46±6.03	324.70±33.48	164.20±14.17¹⁾
50 μg/mL组	98.81±15.55¹⁾	99.52±8.35¹⁾	319.60±15.91	342.70±52.79
F值	8.77	8.07	0.05	8.15
P值	＜0.05	＜0.05	0.95	＜0.05
注：与对照组比较，1)P＜0.05。

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表 3 M2/M1型巨噬细胞相关分子比值

Table 3. The ratio of M2/M1 macrophage-associated molecules

组别	CD206/(CD16/32)		CD369/(CD16/32)
组别	48 h	72 h	48 h	72 h
对照组	0.907±0.002	0.858±0.005	0.024±0.002	0.066±0.002
10 μg/mL组	0.987±0.002¹⁾	0.969±0.002¹⁾	0.008±0.000¹⁾	0.169±0.008¹⁾
50 μg/mL组	0.987±0.002¹⁾	0.962±0.003¹⁾	0.045±0.003¹⁾	0.090±0.002²⁾
F值	1 767.0	953.6	237.5	378.9
P值	＜0.000 1	＜0.000 1	＜0.000 1	＜0.000 1
注：与对照组比较，1)P＜0.000 1，2)P＜0.001。

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