TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

doi:10.19586/j.2095-2341.2021.0163

生物技术进展 ›› 2022, Vol. 12 ›› Issue (1): 142-148.DOI: 10.19586/j.2095-2341.2021.0163

TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

谢月樵(), 郭丹, 王楠, 尤娜, 黄琬玲, 马小彤()

中国医学科学院血液病医院（中国医学科学院血液学研究所），北京协和医学院，实验血液学国家重点实验室，国家血液系统疾病临床医学研究中心，细胞生态海河实验室，天津 300020

收稿日期:2021-10-08 接受日期:2021-11-02 出版日期:2022-01-25 发布日期:2022-01-26
通讯作者: 马小彤
作者简介:谢月樵 E-mail：xieyueqiao@ihcams.ac.cn；
基金资助:
国家自然科学基金项目(82070113)

The Role of TWIST1 in the Proliferation, Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells

Yueqiao XIE(), Dan GUO, Nan WANG, Na YOU, Wanling HUANG, Xiaotong MA()

State Key Laboratory of Experimental Hematology，National Clinical Research Center for Blood Diseases，Haihe Laboratory of Cell Ecosystem，Institute of Hematology & Blood Diseases Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College，Tianjin 300020，China

Received:2021-10-08 Accepted:2021-11-02 Online:2022-01-25 Published:2022-01-26
Contact: Xiaotong MA

摘要/Abstract

摘要：

探讨TWIST1在原代人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）增殖、迁移及体外血管生成中的作用。用有靶向人TWIST1基因shRNA（pLL3.7-shTwist1-GFP）的慢病毒液感染试验组细胞，同时以携带Scramble shRNA的慢病毒液（pLL3.7-shCtrl-GFP）感染对照组细胞，用流式细胞术测定细胞感染效率，实时荧光定量PCR（real-time fluorescent quantitative PCR，qRT-PCR）检测shRNA的基因沉默效率。通过制作细胞生长曲线、Annexin V/7AAD染色流式细胞术、细胞划痕实验、小管形成实验、qRT-PCR检测TWIST1表达降低对HUVECs的增殖、凋亡、迁移、血管形成能力以及血管生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）基因表达的影响。试验组TWIST1基因表达下降为对照组的30%，表明shTWIST1能有效降低TWIST1基因的表达。与对照组相比，敲降TWIST1能明显抑制HUVECs的增殖（P<0.01），诱导细胞凋亡（P<0.05）。试验组HUVECs划痕愈合率、体外生成的血管样结构数目和总小管分支长度均显著低于对照组（P<0.01）；与对照组相比，试验组HUVECs中VEGFR2的表达显著降低（P<0.01）。通过探究HUVECs表达的TWIST1在内皮细胞增殖、存活、迁移和毛细血管样结构的形成中的作用，为TWIST1作为抑制肿瘤血管新生治疗的新靶点提供一定的理论依据。

关键词: 人脐静脉内皮细胞, TWIST1基因, 血管形成, 增殖, 迁移

Abstract:

To explore the function of TWIST1 in the proliferation， migration and angiogenesis of primary human umbilical vein endothelial cells （HUVECs）， the cells in the experimental group were infected with lentiviral solution targeting human TWIST1 gene shRNA （pLL3.7-shTwist1-GFP）， and the cells in the control group were infected with lentiviral solution carrying scramble shRNA （pLL3.7-shCtrl-GFP）， the transfection efficiency was accessed by flow cytometry. TWIST1 mRNA expression level was quantified by real-time fluorescent quantitative PCR （qRT-PCR）. The effects of decreased TWIST1 expression on the proliferation， apoptosis， migration， angiogenesis and vascular growth factor receptor 2 （VEGFR2） gene expression of HUVECs were detected by cell growth curves， flow cytometric analysis of Annexin V/7AAD staining， cell scratch assay， tube formation assay， and qRT-PCR， respectively. TWIST1 gene expression in the experimental group decreased to 30% of that in the control group， indicating that shTWIST1 can effectively reduce TWIST1 gene expression. Compared with the control group， knockdown of TWIST1 could significantly inhibit the proliferation of HUVECs （P<0.01） and induce apoptosis （P<0.05）. The wound healing rate， the number of vascular-like structures and the length of total tubular branches of HUVECs were significantly lower than those of the control group （P<0.01）. Compared with the control group， the expression of VEGFR2 in HUVECs in the experimental group significant decreased（P<0.01）. This study explored the role of TWIST1 expressed by HUVECs in endothelial cell proliferation， survival， migration and the formation of capillary-like structure， and may provide a theoretical basis for TWIST1 as a new target for tumor angiogenesis.

Key words: human umbilical vein endothelial cell （HUVECs）, TWIST1 gene, angiogenesis, proliferation, migration

中图分类号:

R551.1

谢月樵, 郭丹, 王楠, 尤娜, 黄琬玲, 马小彤. TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用[J]. 生物技术进展, 2022, 12(1): 142-148.

Yueqiao XIE, Dan GUO, Nan WANG, Na YOU, Wanling HUANG, Xiaotong MA. The Role of TWIST1 in the Proliferation, Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells[J]. Current Biotechnology, 2022, 12(1): 142-148.

图/表 7

表1 PCR引物序列

Table 1 Sequence of primer in PCR

引物名称	引物序列（5'→3'）
TWIST1	5'⁃GTCCGCAGTCTTACGAGGAG⁃3'
TWIST1	5'⁃TGGAGGACCTGGTAGAGGAA⁃3'
VEGFR2	5'⁃CGGACAGTGGTATGGTTCTTGC⁃3'
VEGFR2	5'⁃GTGGTGTCTGTGTCATCGGAGTG⁃3'
Actin	5'⁃AGTATCCATGAAAATAAGTGGTTACAGG⁃3'
Actin	5'⁃TTTGCAGTACATAATTTACACAGAAGC⁃3'

图1 感染72 h后对照组和试验组HUVECs光镜和荧光显微镜下的观察结果注：放大倍数为×100

Fig.1 The observation results of HUVECs in the control group and the experimental group under light microscope and fluorescence microscope after 72 hours infection

图2 试验组和对照组HUVECs中TWIST1 mRNA水平比较注：**—试验组与对照组相比，差异具有统计学意义（P<0.01）。

Fig.2 Comparison of TWIST1 mRNA levels in HUVECs between experimental group and control group

图3 HUVECs生长曲线和凋亡率A：HUVECs生长曲线；B：HUVECs凋亡率。*表示与对照组相比差异在P<0.05水平具有统计学意义；**表示与对照组相比差异在P<0.01水平具有统计学意义。

Fig.3 Growth curve and apoptosis rate of HUVECs

图4 HUVECs细胞划痕实验和划痕愈合率A：HUVECs细胞划痕实验，放大倍数×100；B：HUVECs划痕愈合率。**表示与对照组相比差异在P<0.01水平具有统计学意义。

Fig.4 Scratch test and scratch healing rate of HUVECs cells

图5 HUVECS血管样结构、总小管分支长度和毛细血管样结构数A：HUVECs血管样结构，放大倍数为×100；B：总小管分支长度和毛细血管样结构数。**表示与对照组相比差异在P<0.01水平具有统计学意义。

Fig.5 Vascular?like structure of HUVECs, total tubular branch length and capillary like structure number

图6 试验组和对照组HUVECs中VEGFR2 mRNA水平比较注：**表示与对照组相比差异在P<0.01水平具有统计学意义。

Fig.6 Comparison of VEGFR2 mRNA levels in HUVECs between experimental group and control group

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TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

The Role of TWIST1 in the Proliferation, Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells

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