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中华细胞与干细胞杂志(电子版) ›› 2026, Vol. 16 ›› Issue (03) : 140 -149. doi: 10.3877/cma.j.issn.2095-1221.2026.03.002

论著

骨髓间充质干细胞外泌体调控TLR4/NF-κB信号通路缓解小鼠子宫内膜异位症研究
姜海珍, 江煜焓, 张丹, 陈晨晨()   
  1. 272000 济宁,山东省济宁医学院附属医院妇科
  • 收稿日期:2026-01-19 出版日期:2026-06-01
  • 通信作者: 陈晨晨

Bone marrow mesenchymal stem cell-derived exosomes alleviate endometriosis in mice by regulating the TLR4/NF-κB signaling pathway

Haizhen Jiang, Yuhan Jiang, Dan Zhang, Chenchen Chen()   

  1. Department of Gynecology, Affiliated Hospital of Jining Medical University, Jining 272000, China
  • Received:2026-01-19 Published:2026-06-01
  • Corresponding author: Chenchen Chen
引用本文:

姜海珍, 江煜焓, 张丹, 陈晨晨. 骨髓间充质干细胞外泌体调控TLR4/NF-κB信号通路缓解小鼠子宫内膜异位症研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2026, 16(03): 140-149.

Haizhen Jiang, Yuhan Jiang, Dan Zhang, Chenchen Chen. Bone marrow mesenchymal stem cell-derived exosomes alleviate endometriosis in mice by regulating the TLR4/NF-κB signaling pathway[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2026, 16(03): 140-149.

目的

探究骨髓间充质干细胞外泌体(BMSC-exo)影响小鼠子宫内膜异位症(EMS)的功能及机制。

方法

小鼠子宫内膜上皮细胞(mEECs)分为BMSC-exo组(25 μg/mL BMSC-exo共孵育)和磷酸盐缓冲溶液(PBS)组(等体积PBS共孵育),EdU法检测细胞增殖率;细胞划痕和Transwell法检测细胞迁移能力。RT-qPCR检测mEECs白细胞介素-1β (IL-1β)、白细胞介素-6 (IL-6)、C-C基序趋化因子配体2 (Ccl-2)和肿瘤坏死因子-α (TNF-α)相对表达量;BALB/c小鼠分为假手术组、EMS模型组和BMSC-exo组(n = 7),EMS组和BMSC-exo组小鼠构建EMS模型,BMSC-exo组小鼠以3 μg/g的剂量每周尾静脉注射1次BMSC-exo (治疗4周),苏木精-伊红染色法(HE)染色观察小鼠EMS病灶组织病理变化;酶联免疫吸附法(ELISA)检测血清和细胞上清中炎症因子IL-1β、IL-6、Ccl-2和TNF-α;Western blot检测小鼠子宫内膜组织TLR4和NF-κB p65表达。两组间比较采用独立样本t检验;多组间比较采用单因素方差分析,组间两两比较采用Dunnett-t检验;不符合正态分布的两组间比较采用Wilcoxon Signed Rank test法;两组间多个时间点同一指标比较采用两因素重复测量方差分析。

结果

与PBS组比较,BMSC-exo组mEECs增殖率[(41.00 ± 3.61)%比(59.67 ± 2.52)%]、细胞划痕愈合率[(22.00 ± 3.00)%比(65.33 ± 5.51)%]、细胞迁移数[(82.00 ± 19.31)比(145.00 ± 9.85)个]降低(P < 0.05),IL-1β (0.18 ± 0.05比1.00 ± 0.01)、IL-6 (0.30 ± 0.09比0.99 ± 0.01)、Ccl-2 (0.27 ± 0.08比1.00 ± 0.02)和TNF-α mRNA相对表达(0.33 ± 0.08比1.00 ± 0.06)均下调(P 均 < 0.01),细胞上清液中IL-1β [(1 083.00 ± 67.68)比(1 507.00 ± 131.10) pg/mL]、IL-6[(878.70 ± 19.50)比(1 127.00 ± 75.59)pg/mL]、Ccl-2[(1 046.00 ± 56.72)比(1 298.00 ± 52.52)pg/mL]和TNF-α浓度[(1 069.00 ± 114.80)比(1 470.00 ± 165.90)pg/mL]降低(P 均 < 0.05)。与假手术组比较,EMS组小鼠子宫内膜病理损伤显著,血清炎症因子IL-1β[(2 034.00 ± 165.50)比(1 083.00 ± 125.80) pg/mL]、IL-6[(3 292.00 ± 232.30)比(1 505.00 ± 126.20)pg/mL]、Ccl-2 [(1 300.00 ± 64.50)比(939.30 ± 56.45)pg/mL]和TNF-α浓度[(545.00 ± 34.70)比(344.00 ± 41.33) pg/mL]增加(P 均 < 0.01),TLR4 (1.44 ± 0.06比0.38 ± 0.06)和NF-κB p65表达(1.18 ± 0.03比0.29 ± 0.04)上调(P 均 < 0.001)。与EMS组比较,BMSC-exo组小鼠EMS组织病灶减少,血清炎症因子IL-1β[(1 488.00 ± 202.50)比(2 034.00 ± 165.50)pg/mL]、IL-6[(2 543.00 ± 317.20)比(3 292.00 ± 232.30)pg/mL]、Ccl-2[(1 058.00 ± 57.95)比(1 300.00 ± 64.50)pg/mL]和TNF-α浓度[(415.00 ± 13.00)比(545.00 ± 34.70)pg/mL]降低(P 均 < 0.05),TLR4 (0.81 ± 0.03比1.44 ± 0.06)和NF-κB p65表达(0.81 ± 0.06比1.18 ± 0.03)下调(P 均 < 0.001)。

结论

BMSC-exo抑制TLR4/NF-κB信号通路缓解小鼠EMS。

Objective

To explore the effects and mechanisms of bone marrow mesenchymal stem cell exosomes (BMSC-exo) on endometriosis (EMS) in mice.

Methods

Mouse endometrial epithelial cells (mEECs) were divided into the BMSC-exo group (co-cultured with 25 μg/mL) and phosphate buffer saline (PBS) group (co-cultured with equal volume of PBS) . The EdU method was used to detect cell proliferation rate. Cell scratch and Transwell assays were employed to assess cell migration ability. RT-qPCR was used to detect the relative expression levels of Interleukin-1β (IL-1β) , interleukin-6 (IL-6) , chemokine 2 (Ccl-2) , and tumor necrosis factor alpha (TNF-α) in mEECs. BALB/c mice were divided into sham-operated, EMS model, and BMSC-exo groups (n = 7) . EMS and BMSC-exo groups were used to construct EMS models. Mice in the BMSC-exo group received weekly tail vein injections of BMSC-exo at a dose of 3 μg/g for 4 weeks. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of EMS lesion tissue in mice. Enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory factors IL-1β, IL-6, Ccl-2, and TNF-α in serum and cell supernatants. Western blot was used to detect the expression of TLR4 and NF-κB p65 in endometrial tissue. Comparisons between two groups were performed using the independent sample t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) , with post-hoc pairwise comparisons conducted using Dunnett's t-test. For data not following a normal distribution, comparisons between two groups were performed using the Wilcoxon signed-rank test. For the analysis of repeated measures factors between two groups, the two-way repeated measures ANOVA was used.

Results

Compared with PBS group, the proliferation rates[ (41.00 ± 3.61) % vs (59.67 ± 2.52) %], cell scratch healing rates [ (22.00 ± 3.00) %vs (65.33 ± 5.51) %], the number of migration cells (82.00 ± 19.31 vs 145.00 ± 9.85) of mEECs, the expression of IL-1β mRNA (0.18 ± 0.05 vs 1.00 ± 0.01) , IL-6 mRNA (0.30 ± 0.09 vs 0.99 ± 0.01) , Ccl-2 mRNA (0.27 ± 0.08 vs 1.00 ± 0.02) , TNF-α mRNA (0.33 ± 0.08 vs 1.00 ± 0.06) and the concentrations of IL-1β[ (1 083.00 ± 67.68) vs (1 507.00 ± 131.10) pg/mL], IL-6[ (878.70 ± 19.50) vs (1 127.00 ± 75.59) pg/mL], Ccl-2 [ (1 046.00 ± 56.72) vs (1 298.00 ± 52.52) pg/mL] and TNF-α[ (1 069.00 ± 114.80) vs (1 470.00 ± 165.90) pg/mL] in cell supernatants in the BMSC-exo group were significantly decreased (P < 0.05) , were significantly decreased (all P < 0.05) . Compared with the sham-operated group, the pathological damageof endometrium in EMS group was significantly, with increased concentrations of serum inflammatory factors IL-1β [ (2 034.00 ± 165.50) vs (1 083.00 ± 125.80) pg/mL], IL-6 [ (3 292.00 ± 232.30) vs (1 505.00 ± 126.20) pg/mL], Ccl-2 [ (1 300.00 ± 64.50) vs (939.30 ± 56.45) pg/mL] and TNF-α [ (545.00 ± 34.70) vs (344.00 ± 41.33)  pg/mL] (all P < 0.01) , as well as a significantly upregulated expression of TLR4 (1.44 ± 0.06 vs 0.38 ± 0.06) and NF-κB p65 (1.18 ± 0.03 vs 0.29 ± 0.04) (all P < 0.001) . Compared with the EMS group, the BMSC-exo group showed a reduction in EMS tissue lesions, with a decrease in the expression of serum inflammatory factors IL-1β [ (1 488.00 ± 202.50) vs (2 034.00 ± 165.50)  pg/mL], IL-6 [ (2 543.00 ± 317.20) vs (3 292.00 ± 232.30) pg/mL], Ccl-2 [ (1 058.00 ± 57.95) vs (1 300.00 ± 64.50) pg/mL], TNF-α [ (415.00 ± 13.00) vs (545.00 ± 34.70) pg/mL] (all P < 0.05) , and TLR4 (0.81 ± 0.03 vs 1.44 ± 0.06) , NF-κB p65 (0.81 ± 0.06 vs 1.18 ± 0.03) expression (all P < 0.001) .

Conclusion

BMSC-exo inhibits TLR4/NF-κB signaling pathway to alleviate EMS in mice.

表1 引物序列信息
图1 倒置相差显微镜下观察BMSC (×100)
图2 流式细胞术检测BMSC注:a ~ e图分别为CD105、CD73、CD90、CD45和CD34表达
图3 BMSC-exo鉴定结果注:a图为透射电镜观察BMSC-exo结构(×10 000);b图为NTA检测BMSC-exo粒径结果;c图为Western blot鉴定BMSC-exo标志蛋白
图4 BMSC-exo对mEECs增殖和迁移能力的影响注:a图为CCK8法检测mEECs增殖能力;b图为荧光显微镜下观察各组细胞增殖率(绿色为EdU染色,蓝色为DAPI染色,×200)及定量比较;c 图为倒置相差显微镜下观察各组细胞划痕愈合率(×200)及定量比较;d图为光学显微镜下观察各组细胞迁移数(结晶紫染色,×200)及定量比较;*P < 0.05,**P < 0.01,***P < 0.001;n = 3
表2 BMSC-exo对mEECs IL-1β、IL-6、Ccl-2和TNF-α mRNA相对表达量的影响( ± s
表3 BMSC-exo对mEECs上清IL-1β、IL-6、Ccl-2和TNF-α分泌的影响( ± s,pg/mL)
图5 BMSC-exo对mEECs的TLR4和NF-κB p65表达的影响注:a图为各组细胞TLR4相对表达量比较;b图为各组细胞NF-κB p65相对表达量比较;***P < 0.001;n = 3
图6 BMSC-exo在EMS小鼠体内分布结果注:Ⅰ、Ⅲ、Ⅴ为不接受DiR标记的BMSC-exo回输的EMS小鼠,作为对照调仪器背景;Ⅱ、Ⅳ、Ⅵ为3只接受DiR标记的BMSC-exo回输的EMS小鼠,观察BMSC-exo在病灶分布
图7 倒置相差显微镜下观察小鼠子宫内膜组织(HE染色,×200)注:a~c分别为假手术组、EMS组、BMSC-exo组;假手术组小鼠子宫内膜上皮细胞完整;EMS模型组小鼠子宫内腺体坏死,出现炎症细胞浸润;BMSC-exo组小鼠子宫内膜组织坏死缓解,病理症状减轻
图8 EMS组和BMSC-exo组小鼠EMS病变组织重量比较注:**P < 0.01,n = 7
表4 各组小鼠血清IL-1β、IL-6、Ccl-2和TNF-α浓度比较( ± s,pg/mL)
图9 各组小鼠子宫内膜组织TLR4和NF-κB相对表达量比较注:a图为各组细胞TLR4相对表达量比较;b图为各组细胞NF-κB p65相对表达量比较;***P < 0.001,n = 3
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[15] 侯建宇, 纪东华. 炎症标志物与外周动脉疾病相关性的研究进展[J/OL]. 中华介入放射学电子杂志, 2026, 14(01): 97-102.
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