Effects of conditioned medium and exosomes of human umbilical cord mesenchymal stem cells on proliferation, migration, invasion, and apoptosis of hepatocellular carcinoma cells

doi:10.12307/2025.012

Abstract

Abstract: BACKGROUND: Mesenchymal stem cells can regulate the tumor microenvironment by secreting extracellular vesicles containing cytokines, growth factors and exosomes for the precise regulation of biological behavior of tumor cells.
OBJECTIVE: To investigate the effects of human umbilical cord-derived mesenchymal stem cell conditioned medium and their released exosomes on the biological properties of hepatocellular carcinoma cells.
METHODS: Human umbilical cord mesenchymal stem cell supernatant was collected, centrifuged and filtered at high speed to obtain human umbilical cord mesenchymal stem cell conditioned medium. Human umbilical cord mesenchymal stem cell supernatant was collected and human umbilical cord mesenchymal stem cell exosomes were extracted by ultra-high speed gradient centrifugation. Human umbilical cord mesenchymal stem cell exosomes were labeled with PKH26 and co-cultured with hepatocellular carcinoma cell MHCC97-H. The uptake of exosomes by MHCC97-H cells was observed by fluorescence microscopy. The effects of human umbilical cord mesenchymal stem cell conditioned medium and human umbilical cord mesenchymal stem cell exosomes on biological functions of hepatocellular carcinoma cells were assessed by the CCK-8 proliferation assay, Transwell migration and invasion assay, and the apoptosis assay.
RESULTS AND CONCLUSION: (1) Human umbilical cord mesenchymal stem cell exosomes could be uptaken by MHCC97-H cells and was mainly distributed in the cytoplasm. (2) After treatment with human umbilical cord mesenchymal stem cell conditioned medium, MHCC97-H cells showed a significant increase in proliferation, migration, and invasion (P < 0.001, P < 0.05, P < 0.01), and a significant decrease in apoptosis (P < 0.001), while after treatment with human umbilical cord mesenchymal stem cell exosomes, MHCC97-H cells showed a decrease in proliferation (P < 0.001) and migration, invasion, and apoptosis were significantly enhanced (P < 0.001). (3) The results indicated that human umbilical cord mesenchymal stem cell conditioned medium had the ability to promote the proliferation, migration, invasion, and inhibit apoptosis of MHCC97-H cells, while human umbilical cord mesenchymal stem cell exosomes had the properties of promoting the migration, invasion and apoptosis of MHCC97-H cells, inhibiting the proliferation.

Key words: hepatocellular carcinoma, human umbilical cord mesenchymal stem cell, exosomes, conditioned medium, proliferation, migration, invasion, apoptosis

CLC Number:

Jin Kai, Tang Ting, Li Meile, Xie Yuan. Effects of conditioned medium and exosomes of human umbilical cord mesenchymal stem cells on proliferation, migration, invasion, and apoptosis of hepatocellular carcinoma cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1350-1355.

Figures/Tables 1

2.1 HUC-MSC-Exo的鉴定结果通过超高速梯度离心法从人脐带间充质干细胞上清液中分离出外泌体，透射电镜观察外泌体呈典型的“茶托状”囊泡结构，见图1A。使用Zeta View粒子追踪器分析表明，外泌体直径大约140 nm，见图1B。Western blot 分析结果表明，HUC-MSC-Exo特异性表达CD9、TSG101和HSP70标志蛋白，但不表达Calnexin蛋白，见图1C。 2.2 MHCC97-H细胞摄取外泌体为了验证HUC-MSC-Exo是否被MHCC97-H细胞摄取，使用PKH26(红色)标记HUC-MSC-Exo，与MHCC97-H细胞在培养箱共同孵育24 h，使用DAPI(蓝色)对细胞核标记，通过荧光显微镜观察发现：MHCC97-H细胞的胞质内有大量红色颗粒物，这表明HUC-MSC-Exo能被MHCC97-H细胞摄取，见图2。 2.3 HUC-MSC-CM及HUC-MSC-Exo对MHCC97-H细胞增殖的影响 CCK-8细胞增殖实验显示，HUC-MSC-CM处理后MHCC97-H细胞增殖显著快于对照组，表明HUC-MSC-CM促进MHCC97-H细胞的增殖，见图3A；而HUC-MSC-Exo处理后MHCC97-H细胞增殖显著慢于对照组，表明HUC-MSC-Exo抑制MHCC97-H细胞的增殖，见图3B。 2.4 HUC-MSC-CM及HUC-MSC-Exo对肝癌细胞迁移和侵袭的影响细胞迁移实验显示，HUC-MSC-CM、HUC-MSC-Exo处理后MHCC97-H 迁移细胞数均显著多于对照组，表明HUC-MSC-CM、HUC-MSC-Exo均促进MHCC97-H细胞的迁移(P < 0.05，P < 0.001)，见图4。细胞侵袭实验显示，HUC-MSC-CM、HUC-MSC-Exo处理后MHCC97-H侵袭细胞数均显著多于对照组，表明HUC-MSC-CM、HUC-MSC-Exo促进MHCC97-H细胞的侵袭(P < 0.01，P < 0.001)，见图4。 2.5 HUC-MSC-CM及HUC-MSC-Exo对肝癌细胞凋亡的影响细胞凋亡实验显示，HUC-MSC-CM处理后MHCC97-H细胞凋亡率显著低于对照组(P < 0.001)，见图5；HUC-MSC-Exo处理后MHCC97-H细胞凋亡率显著高于对照组(P < 0.001)，见图5。 "

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