间充质干细胞与血管内皮细胞间经隧道纳米管线粒体交流对成血管的影响

何云影; 张华; 季平

doi:10.16016/j.2097-0927.202108167

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间充质干细胞与血管内皮细胞间经隧道纳米管线粒体交流对成血管的影响

基础医学 | 更新时间：2026-01-09

- 间充质干细胞与血管内皮细胞间经隧道纳米管线粒体交流对成血管的影响
- Effect of TNTs-mediated mitochondrial transfer between mesenchymal stem cells and endothelial cells on vascularization in vitro
- 陆军军医大学学报 2022年44卷第4期页码：310-319
- 作者机构：
  
  1.400016 重庆，重庆医科大学: 口腔医学院，口腔疾病与生物医学重庆市重点实验室，重庆市高校市级口腔生物医学工程重点实验室
  2.400016 重庆，重庆医科大学: 附属第一医院妇产科
- 作者简介：
  
  [季平，E-mail: jiping@hospital.cqmu.edu.cn JI Ping, E-mail: jiping@hospital.cqmu.edu.cn
- 基金信息：
  
  国家自然科学基金面上项目(82071115)
- DOI：10.16016/j.2097-0927.202108167
  中图分类号： R322.12;R329-33;R329.24
- 收稿：2021-08-27，
  
  修回：2021-09-14，
  
  纸质出版：2022-02-28
- 稿件说明：
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何云影, 张华, 季平. 间充质干细胞与血管内皮细胞间经隧道纳米管线粒体交流对成血管的影响[J]. 陆军军医大学学报, 2022,44(4):310-319.

Yunying HE, Hua ZHANG, Ping JI. Effect of TNTs-mediated mitochondrial transfer between mesenchymal stem cells and endothelial cells on vascularization in vitro[J]. Journal of Army Medical University, 2022, 44(4): 310-319.
何云影, 张华, 季平. 间充质干细胞与血管内皮细胞间经隧道纳米管线粒体交流对成血管的影响[J]. 陆军军医大学学报, 2022,44(4):310-319. DOI： 10.16016/j.2097-0927.202108167.

Yunying HE, Hua ZHANG, Ping JI. Effect of TNTs-mediated mitochondrial transfer between mesenchymal stem cells and endothelial cells on vascularization in vitro[J]. Journal of Army Medical University, 2022, 44(4): 310-319. DOI： 10.16016/j.2097-0927.202108167.

摘要

目的

探讨间充质干细胞(mesenchymal stem cells，MSC)/脐静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)共培养细胞球内的隧道纳米管(tunneling nanotubes，TNTs)对内皮细胞成血管的影响。

方法

采用小管形成实验比较MSC/HUVEC成球共培养组、贴壁共培养组、HUVEC三维培养组和MSC三维培养组的血管生成能力，Western blot和免疫荧光染色检测β-catenin蛋白表达量。以微丝解聚试剂(CytoD)处理MSC/HUVEC共培养细胞球，再次检测CytoD处理组和未处理组的上述指标，并采用荧光标记和场发射扫描电镜验证TNTs结构，采用腺病毒感染的方法观察线粒体交流，采用线粒体膜电位检测评估线粒体状态。

结果

与MSC/HUVEC成球共培养组比较，贴壁共培养组、HUVEC三维培养组和MSC三维培养组的管腔连接交叉点数和小孔数均显著下降(

＜0.05)。β-catenin蛋白免疫荧光染色提示，MSC/HUVEC成球共培养组荧光强度强于贴壁共培养组；Western blot检测结果显示MSC/HUVEC成球共培养组细胞内β-catenin表达显著高于贴壁共培养组(

＜0.05)。鬼笔环肽染色结果显示，共培养球内MSC和HUVEC间存在TNTs(F-actin)。当成球共培养时，MSC胞内观察到来源于HUVEC的线粒体；HUVEC胞内也观察到来源于MSC的线粒体；而上述线粒体交流在CytoD处理组中均减弱；与未处理组比较，CytoD处理组细胞球的线粒体膜电位显著降低(

＜0.05)，HUVEC的增殖速度减慢、迁移范围缩小，细胞球的管腔连接交叉点数和小孔数显著下降(

＜0.05)。CytoD处理组细胞β-catenin蛋白免疫荧光强度弱于未处理组，β-catenin蛋白表达显著低于未处理组(

＜0.05)。

结论

共培养细胞球内，MSC和HUVEC间存在TNTs及其介导的双向线粒体交流，并可能通过β-catenin相关通路影响细胞球成血管能力。

Abstract

Objective

To explore the effect of tunneling nanotubes (TNTs) within mesenchymal stem cells (MSC)/human umbilical vein endothelial cells (HUVEC) co-culture cell spheroids on endothelial cell angiogenesis.

Methods

Pre-vascular network formation assay was used to compare the angiogenic ability among the MSC/HUVEC cell spheroid co-culture group

adherent co-culture group

HUVEC 3D culture group and MSC 3D culture group. The expression of β-catenin protein was detected by Western blotting and immunofluorescence staining. The cell spheroids were treated with microfilament depolymerization reagent (CytoD) as a control group

and the above parameters were tested again in the untreated and treated groups. The structure of TNTs was verified by fluorescent labeling and field emission scanning electron microscopy

mitochondrial communication was observed by adenovirus infection

and the mitochondrial status was assessed by mitochondrial membrane potential assay.

Results

Compared with the MSC/HUVEC cell spheroid co-culture group

the numbers of junctions and meshes were significantly lower in the adherent co-culture group

HUVEC 3D culture group and MSC 3D culture group (

0.05). Immunofluorescence staining for β-catenin protein suggested that the spheroid co-culture group had stronger expression than the adherent co-culture group. The results of Western blotting also showed that the intracellular β-catenin expression in the spheroid co-culture group was significantly higher than that in the adherent co-culture group (

0.05). The presence of TNT (F-actin) between MSC and HUVEC in the co-culture cellular spheroids was shown by phalloidine staining. When the cells were co-cultured as spheroids

the mitochondria derived from HUVEC were observed intracellularly in MSC

and the mitochondria derived from MSC were also observed intracellularly in HUVEC

and the above mitochondrial exchange was diminished in the CytoD-treated group. The mitochondrial membrane potential of the CytoD-treated spheroids was significantly lower than that of the untreated spheroids (

0.05)

and the proliferation rate and migration range of HUVEC were lower and smaller than those of the untreated spheroids. Compared with the untreated cell spheroid group

the numbers of junctions and meshes were significantly lower in the CytoD-treated cell spheroid group (

0.05). The total amount of β-catenin protein was significantly higher in the untreated cell spheroid group compared to the CytoD-treated group (

0.05).

Conclusion

Within the co-culture cell spheroids

there are TNTs and their mediated bidirectional mitochondrial transfer between MSC and HUVEC

and they affect the angiogenic ability of HUVEC within these cellular spheroids probably through β-catenin related pathways.

关键词

Keywords

references

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