外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究

doi:10.12114/j.issn.1007-9572.2023.0768

中国全科医学 ›› 2025, Vol. 28 ›› Issue (08): 962-972.DOI: 10.12114/j.issn.1007-9572.2023.0768

• 论著 • 上一篇

外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究

李梦元, 李冠杉, 韩倩, 李思瑶, 郑舒畅, 辛明蔚, 尹晓丹, 王景尚, 武颖, 何军琴^*()

100026　北京市，首都医科大学附属北京妇产医院/北京妇幼保健院中医科

收稿日期:2023-10-10 修回日期:2024-03-30 出版日期:2025-03-15 发布日期:2025-01-02
通讯作者: 何军琴
作者贡献：
李梦元负责提出研究思路，设计研究方案及论文起草；李冠杉负责实验；韩倩、李思瑶及郑舒畅负责数据的收集与整理；辛明蔚、王景尚、武颖负责绘图与展示；尹晓丹进行论文修订；何军琴负责最终版本修订，对论文负责。
基金资助:
国家自然科学基金面上资助项目(82074477); 北京市自然科学基金面上资助项目(7222268)

Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion

LI Mengyuan, LI Guanshan, HAN Qian, LI Siyao, ZHENG Shuchang, XIN Mingwei, YIN Xiaodan, WANG Jingshang, WU Ying, HE Junqin^*()

Department of Traditional Chinese Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital, Beijing 100026, China

Received:2023-10-10 Revised:2024-03-30 Published:2025-03-15 Online:2025-01-02
Contact: HE Junqin

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摘要/Abstract

摘要： 背景复发性流产（RSA）是常见的生殖障碍性疾病，在胚胎植入过程中，子宫内膜发生上皮间充质转化（EMT），RSA的发生与该过程减弱密切相关。子宫内膜来源外泌体及其携带的miRNA参与细胞间通讯，影响细胞EMT，与RSA关系密切，但具体作用机制尚不明确。目的本研究聚焦于外泌体在细胞EMT中的功能，通过实验验证子宫内膜来源外泌体及其分泌的miRNA-221对RSA小鼠子宫内膜细胞EMT的调控作用。方法 2021年10月—2022年5月，选取SPF级、健康8周龄雌性小鼠24只，随机选取12只作为正常对照组，剩余12只小鼠制作RSA模型，造模结束后雌雄合笼，妊娠第4日处死雌性小鼠，分离和培养子宫内膜上皮细胞并收集外泌体，通过透射电镜和免疫印迹法（WB）进行外泌体鉴定；通过CCK-8、Transwell和流式细胞术检测外泌体干预前后两组细胞增殖、侵袭、迁移能力；细胞EMT相关蛋白[E-cadherin、N-cadherin、Vimentin、基质金属蛋白酶7（MMP-7）、MMP-9]及外泌体标志蛋白（CD63、CD81、TSG101）的表达；外泌体处理后的细胞EMT标志蛋白（CD24- APC、CD44- FITC）的表达；转染miRNA-221模拟物和抑制物分析外泌体源性miRNA-221对小鼠子宫内膜上皮细胞EMT的作用，并通过WB和双荧光素酶报告基因检测进行分析。结果 RSA组小鼠的子宫内膜上皮细胞具有异常的EMT，小鼠子宫内膜上皮细胞能够分泌外泌体。添加外泌体的RSA组细胞增殖率、迁移能力、侵袭能力、CD44+/CD24+表达水平高于细胞上清液RSA组（P<0.05）。与细胞上清液RSA组相比，添加外泌体的RSA组细胞的EMT标志蛋白E-cadherin水平降低，间质样标志蛋白N-cadherin和Vimentin以及基质标志蛋白MMP-7、MMP-9蛋白水平升高（P<0.05）。RSA组miRNA-221表达水平低于正常对照组（P<0.05）。与对照随机miRNA相比，miRNA-221模拟物激活了PI3K-AKT通路、NF-κB通路和p38通路（P<0.05）。与对照随机miRNA相比，miRNA-221模拟物降低了PTEN mRNA的表达水平（P<0.05），而miRNA-221抑制剂转染则增加了PTEN mRNA的表达水平（P<0.05）。结论子宫内膜来源外泌体miRNA-221可通过负向调控Pten基因，激活PI3K-AKT通路，促进子宫内膜上皮细胞EMT，影响RSA妊娠结局。

关键词: 复发性流产, 上皮间充质转化, 外泌体, miRNA-221, Pten基因, PI3K-AKT通路

Abstract:

Background

Recurrent spontaneous abortion (RSA) is a common reproductive disorder closely linked with the weakened epithelial-mesenchymal transition (EMT) in the endometrium during embryo implantation. Endometrium-derived exosomes and miRNAs they transport are linked with RSA via the involvement in cellular communication and EMT, although the specific mechanisms have not yet fully revealed.

Objective

Focusing on the function of exosomes in EMT, this study aims to validate the regulatory role of endometrium-derived exosomes and miRNA-221 they transport in EMT of endometrial cells in RSA mice.

Methods

From October 2021 to May 2022, a total of 24 eight-week-old female mice in the specific pathogen-free (SPF) level were randomly assigned into the control group (n=12) and RSA group (n=12) for modeling. After RSA modeling, male and female mice were housed together. On the 4th day of pregnancy, female mice were sacrificed for isolating endometrial epithelial cells. They were cultured for extracting exosomes, which were identified by transmission electron microscopy and Western blotting. Cell proliferation, invasion, and migration abilities were evaluated using CCK-8 assay, Transwell assay, and fluorescence-activated cell sorting (FACS) before and after exosome interventions. Expression levels of EMT-related genes [E-cadherin, N-cadherin, Vimentin, matrix metalloproteinase 7 (MMP-7), matrix metalloproteinase 9 (MMP-9) ] and exosome marker proteins [CD₆₃, CD₈₁, tumor susceptibility gene 101 (TSG101) ] were measured. Expression levels of EMT marker proteins (CD₂₄^-APC, CD₄₄^- FITC) in exosome-treated cells were analyzed. Transfection of miRNA-221 mimics and inhibitors was performed to analyze the effects of exosome-derived miRNA-221 on EMT of endometrial epithelial cells in RSA mice. Relevant analyses were conducted using Western blot and dual-luciferase reporter assay.

Results

Abnormal EMT was observed in the endometrial epithelial cells of mice in RSA group, and the mouse endometrial epithelial cells were able to secrete exosomes. The proliferation rate, migration ability, invasion ability, and CD₄₄⁺/CD₂₄⁺ ratio of cells in the RSA group treated with exosomes were significantly higher than those measured in cell supernatant of RSA group without exosome intervention (P<0.05). Compared with those in the cell supernatant of RSA group, induction of exosomes significantly downregulated EMT marker protein E-cadherin, but upregulated stromal marker proteins N-cadherin and Vimentin, as well as matrix marker proteins MMP-7 and MMP-9 (P<0.05). The expression level of miRNA-221 in the RSA group was significantly lower than that of the control group (P<0.05). Compared with those transfected with miRNA negative control, transfection of miRNA-221 mimic significantly activated the phosphatidylinositol 3-kinase (PI3K) -AKT pathway, nuclear factor-kappa B (NF-κB) pathway, and p38 pathway (P<0.05), but downregulated mRNA level of PTEN (P<0.05). Transfection of miRNA-221 inhibitor significantly upregulated mRNA level of PTEN (P<0.05) .

Conclusion

Endometrium-derived exosomal miRNA-221 negatively regulates the PTEN gene, activates the PI3K-AKT pathway, promotes EMT in endometrial epithelial cells, and influences the pregnant outcome of RSA mice.

Key words: Recurrent spontaneous abortion, Epithelial-mesenchymal transition, Exosomes, miRNA-221, PTEN gene, PI3K-AKT pathway

中图分类号:

R 714.21

李梦元,李冠杉,韩倩,等. 外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究[J]. 中国全科医学, 2025, 28(08): 962-972. DOI: 10.12114/j.issn.1007-9572.2023.0768.
LI Mengyuan,LI Guanshan,HAN Qian, et al. Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion[J]. Chinese General Practice, 2025, 28(08): 962-972. DOI: 10.12114/j.issn.1007-9572.2023.0768.

图/表 14

参考文献 29

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组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
正常对照组	12	26.33±0.15	43.17±0.70	81.07±0.51	92.87±1.53	126.83±3.68	18.48±0.26	47.10±0.51	85.78±2.80
RSA组	12	26.57±0.55	42.07±0.25	70.77±0.51	81.27±1.50	92.00±2.55	16.34±0.34	37.07±0.45	71.70±1.31
t值		0.707	2.554	24.580	9.381	13.460	8.571	25.550	7.885
P值		0.518	0.063	<0.001	<0.001	<0.001	0.001	<0.001	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
正常对照组	63.10±2.74	29.62±1.06	1.01±0.08	2.74±0.15	2.14±0.31	2.39±0.05	2.60±0.07
RSA组	39.03±2.63	13.11±1.65	1.89±0.05	2.16±0.15	0.79±0.12	1.78±0.14	2.12±0.07
t值	11.110	14.600	16.600	4.626	7.112	7.303	6.785
P值	<0.001	<0.001	<0.001	0.010	0.002	0.002	0.002

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
正常对照组	12	26.33±0.15	43.17±0.70	81.07±0.51	92.87±1.53	126.83±3.68	18.48±0.26	47.10±0.51	85.78±2.80
RSA组	12	26.57±0.55	42.07±0.25	70.77±0.51	81.27±1.50	92.00±2.55	16.34±0.34	37.07±0.45	71.70±1.31
t值		0.707	2.554	24.580	9.381	13.460	8.571	25.550	7.885
P值		0.518	0.063	<0.001	<0.001	<0.001	0.001	<0.001	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
正常对照组	63.10±2.74	29.62±1.06	1.01±0.08	2.74±0.15	2.14±0.31	2.39±0.05	2.60±0.07
RSA组	39.03±2.63	13.11±1.65	1.89±0.05	2.16±0.15	0.79±0.12	1.78±0.14	2.12±0.07
t值	11.110	14.600	16.600	4.626	7.112	7.303	6.785
P值	<0.001	<0.001	<0.001	0.010	0.002	0.002	0.002

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
RSA	12	27.27±0.21	42.57±0.64	70.47±1.42	80.97±1.42	95.57±1.58	17.15±0.05	35.11±6.56	54.85±0.55
RSA+Ex	12	26.59±0.65	42.50±0.60	80.97±1.94	93.30±3.16	129.47±5.23	17.24±0.13	48.89±1.15	64.17±0.43
t值		0.707	0.132	7.560	6.171	10.740	1.103	3.583	23.600
P值		1.721	0.901	0.002	0.004	<0.001	0.332	0.023	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
RSA	26.27±1.91	11.82±1.53	3.47±0.47	2.44±0.15	2.32±0.25	1.92±0.15	1.69±0.10
RSA+Ex	54.33±2.52	30.88±1.46	1.21±0.33	4.45±0.51	3.20±0.08	2.36±0.10	1.96±0.10
t值	15.380	15.610	16.600	6.535	5.855	4.133	3.432
P值	<0.001	<0.001	<0.001	0.003	0.004	0.0145	0.026

组别	例数	细胞增殖					细胞迁移
组别	例数	第0天	第1天	第2天	第3天	第4天	24 h	48 h	72 h
RSA	12	27.27±0.21	42.57±0.64	70.47±1.42	80.97±1.42	95.57±1.58	17.15±0.05	35.11±6.56	54.85±0.55
RSA+Ex	12	26.59±0.65	42.50±0.60	80.97±1.94	93.30±3.16	129.47±5.23	17.24±0.13	48.89±1.15	64.17±0.43
t值		0.707	0.132	7.560	6.171	10.740	1.103	3.583	23.600
P值		1.721	0.901	0.002	0.004	<0.001	0.332	0.023	<0.001
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	EMT标志蛋白			黏附蛋白
组别	细胞侵袭	CD₄₄⁺/CD₂₄⁺	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9
RSA	26.27±1.91	11.82±1.53	3.47±0.47	2.44±0.15	2.32±0.25	1.92±0.15	1.69±0.10
RSA+Ex	54.33±2.52	30.88±1.46	1.21±0.33	4.45±0.51	3.20±0.08	2.36±0.10	1.96±0.10
t值	15.380	15.610	16.600	6.535	5.855	4.133	3.432
P值	<0.001	<0.001	<0.001	0.003	0.004	0.0145	0.026

组别	例数	miRNA-133a	miRNA-158-5P	miRNA-221	miRNA-720	miRNA-365	miRNA-215	miRNA-34a
正常对照组	12	0.92±0.06	0.93±0.07	5.07±0.10	0.92±0.06	0.92±0.05	0.97±0.12	0.92±0.06
RSA组	12	1.18±0.04	2.10±0.12	1.05±0.14	0.49±0.03	1.13±0.07	5.65±0.46	0.42±0.03
t值		6.390	14.000	40.550	11.970	4.209	17.190	13.690
P值		0.003	<0.001	<0.001	<0.001	0.014	<0.001	<0.001

外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究

Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion

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组别	例数	E-cadherin	N-cadherin	Vimentin	MMP-7	MMP-9	P-IKKα/β	IKKα/β
Scrambled miR	12	0.85±0.04	0.63±0.05	0.69±0.06	1.04±0.08	0.74±0.04	1.73±0.15	1.02±0.06
miR-221 mimic	12	0.67±0.05	1.06±0.06	0.93±0.06	1.20±0.03	1.71±0.17	4.13±0.08	1.01±0.06
miR-221 inhibi	12	1.64±0.10	0.51±0.02	0.46±0.02	0.48±0.07	0.40±0.06	1.22±0.12	1.01±0.04
F值		127.700	114.100	65.790	104.000	121.400	61.820	0.042
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.958
组别	P-p38	p38	IL-6	EGFR	P-Akt	Akt	Pten
Scrambled miR	0.36±0.12	0.75±0.05	0.96±0.01	1.48±0.06	3.15±0.03	1.56±0.12	1.46±0.02
miR-221 mimic	1.77±0.12	0.81±0.04	0.92±0.03	1.46±0.06	2.40±0.25	1.45±0.31	1.38±0.17
miR-221 inhibi	1.43±0.10	0.80±0.05	0.93±0.01	1.45±0.05	2.56±0.27	1.62±0.21	3.54±0.21
F值	131.100	1.314	2.603	0.531	10.270	0.411	177.100
P值	<0.001	0.336	0.154	0.613	0.011	0.680	<0.001

组别	例数	P-Akt	Akt	Pten
Control	12	1.76±0.08	1.38±0.15	2.54±0.12
SF1670	12	2.49±0.08	1.35±0.13	1.35±0.05
PF-04691502	12	3.53±0.17	1.40±0.04	1.56±0.17
F值		163.10	0.18	79.48
P值		<0.001	0.840	<0.001

组别	例数	细胞侵袭能力	E-cadherin	N-cadherin	Vimentin	P-Akt	Akt
Control	12	40.90±0.56	3.16±0.09	2.41±0.11	2.62±0.23	3.54±0.26	1.30±0.13
miR-221 mimic	12	76.53±1.98	5.11±0.04	2.25±0.12	0.82±0.10	0.74±0.10	1.10±0.06
miR-221 mimic+PF-04691502	12	55.26±2.50	4.24±0.06	4.76±0.23	3.17±0.20	2.33±0.15	0.86±0.06
F值		276.20	364.90	229.00	129.80	179.30	18.92
P值		<0.001	<0.001	<0.001	<0.001	<0.001	0.003

[1]	柴晏, 赵玉青, 郭旭男, 王东英, 边云飞. 冠状动脉粥样硬化性心脏病患者心外膜脂肪组织的生物信息学研究[J]. 中国全科医学, 2023, 26(08): 939-950.
[2]	刘滔滔, 李天容, 王雪, 陈家蒙, 帅芝琴, 李利生, 徐尚福. 纳米颗粒和外泌体靶向载药系统诊治动脉粥样硬化的机遇与挑战[J]. 中国全科医学, 2023, 26(08): 903-910.
[3]	张清华，潘静，姚丽艳. 复发性流产患者病因构成分析[J]. 中国全科医学, 2020, 23(14): 1760-1764.
[4]	冯晓玲，常卓，赵小萱，王炜，李雯雯，陈璐. 不明原因复发性流产患者的D型人格与中医证候分布研究[J]. 中国全科医学, 2018, 21(34): 4227-4231.
[5]	贺继刚，李贝贝，韩金秀，李洪荣，撒亚莲，严丹，杨晓华. 过表达GATA-4小鼠骨髓间充质干细胞通过分泌外泌体增强其抗凋亡能力的研究[J]. 中国全科医学, 2018, 21(24): 2961-2966.
[6]	贾慧琼1，冷雪芹2*，李燕3，鲁志刚4. PTEN和低氧诱导因子-1α在蒙古族、汉族胃癌患者中的表达及临床意义[J]. 中国全科医学, 2017, 20(10): 1214-1219.

组别	例数	细胞增殖				细胞迁移			细胞侵袭	CD₄₄⁺/CD₂₄⁺
组别	例数	第1天	第2天	第3天	第4天	24 h	48 h	72 h	细胞侵袭	CD₄₄⁺/CD₂₄⁺
Scrambled miR	12	18.67±0.67	36.90±0.40	57.53±0.65	79.63±3.52	19.11±0.14	45.16±0.96	66.2±0.40	31.73±1.53	21.64±1.09
miR-221 mimic	12	18.07±0.67	38.20±0.30	73.17±4.14	102.13±3.00	20.23±0.17	52.53±1.02	74.29±1.04	60.36±2.54	32.83±1.53
miR-221 inhibi	12	18.07±0.38	37.23±0.25	40.93±1.72	70.73±8.11	17.22±0.15	36.06±0.22	58.50±0.87	25.51±3.90	11.82±1.53
F值		1.067	13.100	113.900	27.040	296.300	303.900	282.100	129.500	169.900
P值		0.401	0.006	<0.001	0.001	<0.001	<0.001	<0.001	<0.001	<0.001

组别	例数	细胞增殖				细胞迁移			细胞侵袭	CD₄₄⁺/CD₂₄⁺
组别	例数	第1天	第2天	第3天	第4天	24 h	48 h	72 h	细胞侵袭	CD₄₄⁺/CD₂₄⁺
Scrambled miR	12	18.67±0.67	36.90±0.40	57.53±0.65	79.63±3.52	19.11±0.14	45.16±0.96	66.2±0.40	31.73±1.53	21.64±1.09
miR-221 mimic	12	18.07±0.67	38.20±0.30	73.17±4.14	102.13±3.00	20.23±0.17	52.53±1.02	74.29±1.04	60.36±2.54	32.83±1.53
miR-221 inhibi	12	18.07±0.38	37.23±0.25	40.93±1.72	70.73±8.11	17.22±0.15	36.06±0.22	58.50±0.87	25.51±3.90	11.82±1.53
F值		1.067	13.100	113.900	27.040	296.300	303.900	282.100	129.500	169.900
P值		0.401	0.006	<0.001	0.001	<0.001	<0.001	<0.001	<0.001	<0.001

分类	组合1	组合2	组合3	组合4	组合5	组合6	组合7	组合8	F值	P值
miR-221 mimic	+	-	-	+	-	-	-	-
miR-221 inhibi	-	+	-	-	+	-	-	-
Scrambled miR	-	-	+	-	-	+	-	-
Pten 3' UTR-WT	+	+	+	-	-	-	+	-
Pten 3' UTR-Mut	-	-	-	+	+	+	-	+
数值	4.28±0.12^a	26.11±0.21^a	14.75±0.53	13.53±0.07	13.14±0.40	13.64±0.46	13.53±0.31	13.52±0.07	1 024	<0.001

分类	组合1	组合2	组合3	组合4	组合5	组合6	组合7	组合8	F值	P值
miR-221 mimic	+	-	-	+	-	-	-	-
miR-221 inhibi	-	+	-	-	+	-	-	-
Scrambled miR	-	-	+	-	-	+	-	-
Pten 3' UTR-WT	+	+	+	-	-	-	+	-
Pten 3' UTR-Mut	-	-	-	+	+	+	-	+
数值	4.28±0.12^a	26.11±0.21^a	14.75±0.53	13.53±0.07	13.14±0.40	13.64±0.46	13.53±0.31	13.52±0.07	1 024	<0.001