抗血管生成纳米多肽通过抑制VEGF和TIE-2双信号通路延缓肾透明细胞癌进展的研究

doi:10.11904/j.issn.1002-3070.2025.02.007

摘要/Abstract

摘要： 目的设计并合成一种同时抑制血管内皮生长因子(vascular endothelial growth factor,VEGF)/具有免疫球蛋白样及表皮生长因子样结构域的酪氨酸蛋白激酶-2(tyrosine kinase with immunoglobulin-like and EGF-like domains-2,TIE-2)的靶向纳米多肽(VEGF/TIE-2 targeted nanopeptides,VTN),探索其对肾透明细胞癌(renal clear cell carcinoma,ccRCC)血管生成的抑制作用。方法采用固相肽合成技术制备VTN及不可自组装对照VTN-C,并利用电喷雾电离质谱法(electrospray ionization mass spectrometry,ESI-MS)对VTN和VTN-C的分子结构进行分析。通过CCK-8法评估VTN对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)活力的影响;通过细胞划痕实验、Transwell侵袭实验及血管形成实验分别检测VTN对HUVEC侵袭、迁移和血管形成的影响;通过Western blot实验检测VTN对VEGF和TIE-2信号通路下游蛋白磷酸化的影响;建立786-O荷瘤小鼠模型,通过动物实验观察VTN对体内肿瘤血管生成和肿瘤进展的影响。结果 ESI-MS显示,合成的VTN和VTN-C主要电荷状态峰均指向同一分子量,且与相应理论分子质量高度一致,免疫荧光显示VTN与VEGF和TIE-2共定位,VTN联合MMP-2能显著抑制HUVEC活力(P＜0.001),VTN组细胞侵袭率、划痕愈合率较PBS组分别降低(78.30±1.35)%和(37.09±3.49)%(P＜0.001),血管形成实验表明VTN能显著抑制HUVEC细胞血管形成能力(P＜0.001),Western blot显示VTN显著抑制Akt和ERK磷酸化(P＜0.001)。体内实验表明,VTN组肿瘤体积较对照组减小(87.16±1.30)%,CD31阳性面积降低(P＜0.01)。结论 VTN通过抑制VEGF和TIE-2信号通路,下调Akt和ERK磷酸化,显著阻断ccRCC血管生成,延缓肿瘤进展。

关键词: 肾透明细胞癌, 纳米多肽, 血管生成, 血管内皮生长因子, 具有免疫球蛋白样及表皮生长因子样结构域的酪氨酸蛋白激酶-2

Abstract: Objective A targeted nanopeptides(VEGF/TIE-2 targeted nanopeptides,VTN)that simultaneously inhibits vascular endothelial growth factor(VEGF)/ tyrosine kinase with immunoglobulin-like and EGF-like domains-2(TIE-2)signaling pathways were designed and synthesized,and explore its inhibitory effect on angiogenesis in renal clear cell carcinoma(ccRCC). Methods VTN and non-self-assembling control VTN-C were prepared by solid-phase peptide synthesis technology,and the molecular structures of VTN and VTN-C were analyzed by electrospray ionization mass spectrometry(ESI-MS).The CCK-8 method was used to evaluate the effect of VTN on the cell viability of human umbilical vein endothelial cells(HUVEC).The cell scratch assay,Transwell invasion assay and angiogenesis assay were used to detect the inhibitory effects of VTN on migration,invasion and angiogenesis of HUVEC.Western blot was used to detect the effect of VTN on the phosphorylation of downstream proteins of VEGF and TIE-2 signaling pathways.A 786-O cell mouse model was established,and the effects of VTN on tumor angiogenesis and tumor progression were observed through animal experiments. Results ESI-MS showed that the main charge state peaks of both synthesized VTN and VVTN-C pointed to the same molecular weight,which was highly consistent with the corresponding theoretical molecular mass.Immunofluorescence showed that VTN co-localized with VEGF and TIE-2.VTN combined with MMP-2 could significantly inhibit the activity of HUVEC(P＜0.001).The cell invasion rate and scratch closure rate in the VTN group were reduced by(78.30±1.35)% and(37.09±3.49)% compared those in the PBS group,respectively(P＜0.001).Angiogenesis experiments showed that VTN could significantly inhibit the angiogenesis of HUVEC(P＜0.001).Western blot showed that VTN significantly inhibited the phosphorylation of Akt and ERK(P＜0.001).The results from animal experiments showed that tumor volume in the VTN group was decreased by(87.16±1.30)% compared with the control group,and the CD31-positive area was reduced(P＜0.001). Conclusion VTN significantly blocks ccRCC angiogenesis and delays tumor progression by inhibiting VEGF and TIE-2 signaling pathways and downregulating Akt and ERK phosphorylation.

Key words: Renal clear cell carcinoma, Nanopeptide, Angiogenesis, Vascular endothelial growth factor, Tyrosine protein kinase with immunoglobulin-like and epidermal growth factor-like domains-2

中图分类号:

R737.1

刘攀, 孔斌, 臧佳慧, 王新月, 孙月, 王璐, 徐万海. 抗血管生成纳米多肽通过抑制VEGF和TIE-2双信号通路延缓肾透明细胞癌进展的研究[J]. 实用肿瘤学杂志, 2025, 39(2): 116-125.

LIU Pan, KONG Bin, ZANG Jiahui, WANG Xinyue, SUN Yue, WANG Lu, XU Wanhai. The effect of anti-angiogenic nanopeptides on delaying the progression in clear cell renal carcinoma by inhibiting the dual signaling pathways of VEGF and TIE-2[J]. Journal of Practical Oncology, 2025, 39(2): 116-125.

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