兰州大学机构库 >基础医学院
黄芪多糖在肿瘤源性胞外囊泡介导胃癌血道转移中的保护作用及机制研究
Alternative TitleThe Protective Role and Mechanism of Astragalus Polysaccharides in Tumor-derived Extracellular Vesicles Induced Hematogenous Metastasis of Gastric Cancer
王敏
Subtype博士
Thesis Advisor李敏
2023-09-05
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline中西医结合临床
Keyword肿瘤源性胞外囊泡 Tumor-derived Extracellular Vesicles 黄芪多糖 Astragalus polysaccharides 内皮屏障 Endothelial barrier 胃癌 Gastric cancer 血道转移 Hematogenous Metastasis
Abstract

背景:血道转移是恶性肿瘤患者的主要致死原因,血管内皮屏障完整性是影响肿瘤细胞跨内皮迁移、导致肿瘤血道转移的关键。肿瘤源性胞外囊泡(Tumor-derived Extracellular Vesicles, TEVs)作为肿瘤细胞释放的具有脂质双层膜结构的纳米级囊泡,通过参与肿瘤微环境、免疫逃避、血管生成等环节,调控肿瘤的进展。临床研究发现肿瘤患者外周血TEVs水平随肿瘤进展增高,但其能否通过影响血管内皮通透性促进肿瘤转移,目前尚不明确。

前期的合作研究发现脑外伤时脑源性微粒能破坏血脑屏障进入外周循环,并在体内外实验中诱导血管内皮高通透性,促进EVs的清除可保护由缺血性损伤而导致的内皮完整性破坏。因此我们推测TEVs可能通过影响血管内皮通透性促进胃癌的血道转移。

肿瘤转移机制研究与肿瘤治疗密不可分。黄芪多糖(Astragalus polysaccharides, APS)作为甘肃省道地药材-黄芪的主要成分,研究发现APS通过抑制肿瘤细胞增殖、促进凋亡、增强机体免疫功能等途径发挥抗肿瘤作用,但黄芪多糖是否还存在其他抗肿瘤机制尚不可知。研究人员发现APS在心脑血管疾病中对血管内皮损伤具有保护作用,那么在肿瘤血道转移进程中,血浆中某些因素如TEVs可能导致靶器官血管内皮屏障损伤,APS是否能够发挥内皮保护作用?这为APS抗肿瘤转移机制研究提供了新方向。

目的:以我国西部高发胃癌为研究对象,探讨TEVs是否通过激活内皮细胞、增加血管内皮通透性、增强胃癌细胞黏附并跨内皮迁移能力,促进肿瘤转移;阐明VEGFA-VEGFR2-Src-VE-cadherin信号通路与TEVs介导内皮屏障损伤的关系;探讨黄芪多糖在TEVs介导胃癌血道转移中的作用,同时运用网络药理学方法筛选APS的活性成分及作用靶标,进一步验证APS的可能作用机制。

方法:

1. 收集胃癌患者血液样本,应用电镜、流式细胞术、ELISA检测等方法分析胃癌患者血浆TEVs水平与胃癌分化程度、转移及VWF含量的相关性。   

2. 建立小鼠肿瘤模型,尾静脉单独或联合注射TEVs和乳凝集素,通过流式细胞术、纳米颗粒跟踪、组织学观察、免疫组化等方法,分析荷瘤小鼠血浆TEVs水平与肿瘤大小、转移的关系,阐明TEVs体内对肿瘤细胞肺转移的影响。

3. 体外分离提取TEVs,电镜及NTA等方法鉴定免疫表型和形态特征;激光共聚焦及流式细胞术检测TEVs与内皮细胞的作用方式;跨内皮电阻测定、Transwell模型及小鼠体内实验检测TEVs对内皮屏障通透性及肿瘤细胞跨内皮迁移的影响;应用扫描电镜、免疫荧光、ELISA及In-Cell Western等方法检测TEVs作用后内皮细胞形态、VWF释放及内皮表面粘附分子CD31和骨架蛋白F-actin的表达,分析TEVs作用对VEGFA-VEGFR2-Src-VE-cadherin通路的影响。

4. 建立小鼠实验性肺转移模型,通过腹腔注射APS,应用流式、组织学观察、免疫组化及ELISA等方法,检测荷瘤小鼠外周血TEVs、VWF水平及肺组织荷瘤情况,检测APS对荷瘤小鼠体重、免疫器官、肺转移灶免疫细胞浸润及肝脏巨噬细胞数量的影响,分析APS对TEVs介导荷瘤小鼠肿瘤肺转移的作用。

5. APS体外作用胃癌细胞,流式细胞术检测TEVs释放的数量。APS作用血管内皮细胞,应用流式、共聚焦、ELSA、体内外通透性实验、免疫荧光及In-Cell Western等方法,分析APS对TEVs与内皮相互作用、内皮细胞通透性、肿瘤细胞粘附并跨迁移及内皮细胞激活的影响,阐明APS对VEGFA-VEGFR2 通路的作用。APS体外作用小鼠巨噬细胞,检测对巨噬细胞骨架蛋白、吞噬能力及清除TEVs的影响。

6. 运用中药网络药理学方法,筛选黄芪多糖作用胃癌转移的主要活性成分和潜在靶点,对其核心基因进行生物学分析。

结果:

1. 胃癌患者外周血总EVs及TEVs水平较健康受试者均显著增高;低分化组明显高于中分化、高分化、上皮内肿瘤患者及健康受试者;转移组明显高于未转移组;胃癌患者外周血TEVs水平与性别、年龄无相关性,与肿瘤组织的分化程度呈负相关,与肿瘤转移及血浆VWF水平呈正相关。

2. 皮下移植瘤小鼠血浆TEVs水平随肿瘤增大而增高,发生肺转移时明显高于未转移组。实验性肺转移模型小鼠TEVs组肺表面结节数、肺重及肺荷瘤率明显高于对照组,血浆和肺组织VWF表达增加;乳凝集素作用后,外周血TEVs的水平降低,肺转移减少。

3. 体外提取的TEVs作用24h被内皮细胞摄入,Dynasore部分抑制内皮细胞摄入TEVs;TEVs作用后内皮屏障通透性增加、胃癌细胞跨内皮迁移增加,内皮细胞激活,VWF释放增多,细胞骨架收缩,细胞形态改变,粘附分子CD31表达降低,TEVs作用能激活内皮细胞VEGFA-VEGFR2-Src-VE-cadherin通路。

4. 黄芪多糖体内显著降低由 TEVs 介导的小鼠肿瘤细胞肺转移,并降低荷瘤小鼠血浆TEVs与VWF水平;APS能部分改善由MFC及TEVs造成的体重下降,提升脾指数和胸腺指数,增强CD8+T淋巴细胞、CD20+B淋巴细胞和CD68+巨噬细胞对肺转移灶的浸润,增加肝脏巨噬细胞数量。

5. 黄芪多糖体外能减少胃癌细胞产生TEVs。APS联合作用内皮细胞时可降低TEVs介导的内皮屏障通透性增加、胃癌细胞跨内皮迁移增多,部分抑制TEVs介导的内皮细胞激活及VEGFA-VEGFR2-Src-VE-cadherin通路活化。APS体外促进小鼠巨噬细胞细胞骨架蛋白重排,增强其吞噬能力并增加对TEVs的吞噬。

6. 中药网络药理学发现黄芪多糖活性成分11个,潜在作用靶标299个,胃癌转移相关靶标9875个,APS作用胃癌转移的核心靶标130个;GO和KEGG分析进一步证实黄芪多糖能够通过细胞外囊泡及VEGFA/VEGFR2 通路发挥抗胃癌转移作用。

结论:

1. 胃癌患者血浆TEVs水平随肿瘤进展升高提示其参与胃癌进展。TEVs通过激活血管内皮细胞,刺激VWF释放增加,增强肿瘤细胞对内皮的粘附;通过影响细胞骨架蛋白及细胞表面粘附分子的表达,增加血管内皮通透性,增强肿瘤细胞跨内皮迁移,进而促进肿瘤血道转移;TEVs介导内皮屏障损伤与激活VEGFA-VEGFR2-Src-VE-cadherin通路,降低VE-cadherin表达密切相关。

2. 黄芪多糖在TEVs介导的肿瘤血道转移中起保护作用,增强抗肿瘤免疫、减少TEVs的产生并促进巨噬细胞对TEVs的清除、抑制TEVs介导的内皮细胞激活及VEGFA-VEGFR2通路活化,可能是其抗肿瘤转移的新机制。

3. 中药网络药理学研究证实黄芪多糖能够通过细胞外囊泡及VEGFA/ VEGFR2通路,发挥抗胃癌转移作用;黄芪多糖对胃癌转移的调控是一个多靶点-多通路-多效应的分子网络模式。

Other Abstract

Background Hematogenous metastasis is the main cause of death in malignant tumor. The integrity of the vascular endothelium barrier is the key to affecting the transendothelial migration of tumor cells and leading to the blood metastasis of tumor. Tumor-derived Extracellular Vesicles (TEVs), as nanoscale lipid bilayer membranes released by tumor cells, regulate tumor progression by participating in tumor microenvironment, immune escape, and angiogenesis. Clinical studies have found that the level of TEVs in peripheral blood of cancer patients increases with tumor progression, but whether it can promote tumor metastasis by affecting vascular endothelial permeability is still unclear.

Previous collaborative studies have found that brain-derived microparticles can break the blood-brain barrier and enter the peripheral circulation during brain injury, induce high vascular endothelial permeability in vivo and in vitro experiments, and promote EVs clearance to protect the endothelial integrity damage caused by ischemic injury. Therefore, we speculated that TEVs might promote blood metastasis of gastric cancer by inducing vascular endothelial permeability.

The study of tumor metastasis mechanism is closely related to tumor therapy. Astragalus polysaccharides (APS), as the main component of Astragalus, played an anti-tumor role by inhibiting the proliferation of tumor cells, promoting apoptosis and enhancing immune function. It is not known whether Astragalus polysaccharides have other anti-tumor mechanisms. Researchers have found that APS has a protective effect on vascular endothelial damage in cardiovascular and cerebrovascular diseases. In the process of tumor metastasis, some factors in plasma, such as TEVs, may cause vascular endothelial barrier damage of target organs. APS may play a role in endothelial protection, which provides a new direction for the study of the anti-tumor metastasis mechanism of APS.

Objective This research project intends to use high-risk gastric cancer in western China as a research object to investigate whether TEVs activate endothelial cells, increase vascular endothelial permeability, enhance gastric cancer cell adhesion and transendothelial migration, and promote tumor metastasis. It is elucidated the relationship between VEGFA-VEGFR2-Src-VE-cadherin signaling pathway and TEVs mediating endothelial barrier injury, the role and possible mechanism of APS in TEVs mediating hematogenous metastasis of gastric cancer is clarifying.

Methods

1. Peripheral blood was collected from patients with gastric cancer and healthy subjects. Plasma EVs and TEVs were detected by flow cytometry, plasma VWF levels were measured by ELISA. The correlation of the levels of plasma TEVs with differentiation, metastasis, and VWF levels in gastric cancer were analyzed by correlation analysis.

2. Mouse gastric cancer subcutaneously transplanted and metastasis model were established. Flow cytometry was used to detect plasma TEVs content in tumor-bearing mice, and the relationship between TEVs level with tumor size and metastasis was analyzed by correlation analysis. TEVs were injected into tail vein of tumor-bearing mice to increase the content, and Lactadherin were injected to reduce plasma TEVs levels. Pulmonary surface nodules, weights, and lung metastasis areas were observed. VWF was detected in plasma and lung tissues. We explored the role of TEVs in hematogenous metastasis of gastric cancer.

3.TEVs were isolated and obtained in vitro, which phenotype and morphology were identified by flow cytometry and electron microscopy. Laser confocal and flow cytometry were used to examine the relationship of TEVs and endothelial cells. Transendothelial electrical resistance assay, Transwell model and Mouse transplanted tumor were used to detect the effects of TEVs on endothelial barrier permeability and tumor cell transendothelial migration. Scanning electron microscopy, ELISA and immunofluorescence were used to detect the morphology of endothelial cells, VWF release, and the expression of adhesion molecules CD31 and F-actin on the endothelial surface after TEVs treatment. The effect of TEVs on the VEGFA- VEGFR2-Src-VE-cadherin pathway were detected by In-Cell Western, ELISA and immunofluorescence methods.

4. Mouse metastasis model was established. The changes of lung metastasis and immune organs in tumor-bearing mice were detected by intraperitoneal injection of APS, and the related role of APS in TEVs-mediated hematogenous metastasis was analyzed.

5. By means of in vitro experiments, we examined the effects of APS on gastric cancer cells releasing TEVs.We also researched the effects of APS on TEVs mediated endothelial interaction, permeability, tumor cell adhesion and transmigration, endothelial cell activation, and VEGFA-VEGFR2 pathway. In addition, APS was used on macrophages in vitro to detect the effects on macrophage cytoskeleton protein, phagocytosis and TEVs clearance.

6.The main active components and potential targets of Astragalus polysaccharide on gastric cancer metastasis were screened by network pharmacology, and its core genes were analyzed.

Results

 1. The levels of total EVs and TEVs in peripheral blood of gastric cancer patients were significantly higher than those in healthy subjects. The levels of peripheral blood TEVs in patients with poorly differentiated gastric cancer were significantly higher than those in moderately differentiated, well-differentiated, intraepithelial tumors, and healthy subjects. TEVs levels in peripheral blood of gastric cancer patients with metastasis were significantly higher than those without metastasis. TEVs levels in peripheral blood of gastric cancer patients were not correlated with gender and age, but were negatively correlated with tumor differentiation, and were positively correlated with tumor metastasis and plasma VWF levels.

2. The levels of plasma TEVs in tumor-bearing mice increased with the increase of tumor size. And the levels of plasma TEVs in metastatic tumor-bearing mice were significantly higher than those in non-metastatic group. The surface pulmonary nodule, lung weight, and tumor-bearing areas in TEVs group were significantly higher than control group, and VWF expression in the plasma and lung tissues was increased. The level of TEVs in peripheral blood, tumor-bearing areas of lung tissue, and the expression of VWF decreased after Lactadherin treatment.

3. TEVs were isolated and acted on endothelial cells for 24h in vitro, then TEVs were intaked by endothelial cells. Dynasore partially inhibited the action of endothelial cells and TEVs. The permeability of endothelial barrier and the transendothelial migration of gastric cancer cells increased after TEVs were treated. Endothelial activation, VWF release, cytoskeleton contraction, cell morphology changed, and CD31 expression decreased after TEVs were treated. TEVs could activate the VEGFA-VEGFR2-Src-VE-cadherin pathway.

4. APS could reduce the increase of lung metastasis mediated by TEVs, and reduce plasma TEVs and VWF levels. APS could also partially improve the weight loss caused by MFC and TEVs, increase the spleen index and thymus index, increase the infiltration of CD8+T cells, CD20+B cells and CD68+ macrophages to metastasis, and increase the number of liver macrophages.

5. APS could reduce the production of TEVs and TEVs-mediated increase of endothelial barrier permeability and increased transendothelial migration of gastric cancer cells, partially inhibited the activation of TEVs-mediated endothelial cells and VEGFA-VEGFR2 pathway. In addition, APS promoted the cytoskeletal protein rearrangement of mouse macrophages, enhanced their phagocytosis and increased the phagocytosis of TEVs.

6. The network pharmacology of traditional Chinese medicine found 11 active components of APS, 299 potential targets, 9875 targets related to gastric cancer metastasis, and 130 core targets of APS for gastric cancer metastasis. GO and KEGG analysis confirmed that APS could play an anti-metastasis role in gastric cancer through extracellular vesicles and VEGFA/VEGFR2 pathway.

Conclusion

1. Level of plasma TEVs in patients with gastric cancer increased with tumor progression, suggesting that TEVs was involved in gastric cancer progression. TEVs can promote the hematogenous metastasis by activating vascular endothelial cells, increasing the permeability of endothelial barrier, enhancing the adherence of tumor cells to the endothelium and transendothelial migration. TEVs-mediated endothelial barrier damage is closely related to activation of VEGFA-VEGFR2-Src-VE-cadherin pathway and decrease of VE-cadherin expression.

2. Astragalus polysaccharides plays a protective role in TEVs-mediated tumor metastasis. Enhancing anti-tumor immunity, reducing the production of TEVs, promoting the clearance of TEVs by macrophages, inhibiting TEVs-mediated endothelial cell activation and VEGFA-VEGFR2 pathway activation, which may be  new mechanisms of its anti-tumor metastasis.

3.Traditional Chinese medicine network pharmacological studies have confirmed that APS can play an anti-metastasis role in gastric cancer through extracellular vesicles and VEGFA/ VEGFR2 pathway. The regulation of APS on gastric cancer metastasis is a multitarget-multipathway-multieffect molecular network model.

Subject Area中医药抗肿瘤转移机制研究
MOST Discipline Catalogue医学 - 中西医结合 - 中西医结合临床
URL查看原文
Language中文
Other Code262010_120120900990
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538037
Collection基础医学院
Affiliation
兰州大学基础医学院
Recommended Citation
GB/T 7714
王敏. 黄芪多糖在肿瘤源性胞外囊泡介导胃癌血道转移中的保护作用及机制研究[D]. 兰州. 兰州大学,2023.
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