The roles of LMO4 in endothelial cells differentiation and angiogenesis from murine embryonic stem cells
10.19405/j.cnki.issn1000-1492.2024.01.001.
- Author:
Minghua Xiang
1
;
Zhenzhen Tu
2
;
Yue Wang
3
;
Haisheng Zhou
4
Author Information
1. Dept of Applied Physics，School of Biomedical Engineering，Anhui Medical University，Hefei 230032
2. Institute of Clinical Immunology，The First Affiliated Hospital of Anhui Medical University，Hefei 230022
3. Clinical College of Anhui Medical University，Hefei 230031
4. Dept of Biochemistry，School of Basic Medical Sciences，Anhui Medical University，Hefei 230032
- Publication Type:Journal Article
- Keywords:
mouse embryonic stem cell;
embryoid body;
hemangioblast;
endothelial cell;
angiogenesis
- From:
Acta Universitatis Medicinalis Anhui
2024;59(1):1-7
- CountryChina
- Language:Chinese
-
Abstract:
Objective :To examine the role of LMO4 in the regulation of endothelial cell differentiation and angio- genesis in murine embryonic stem cells (mESC) ．
Methods :Mouse Lmo4 cDNA was obtained from MEL cells by using the reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into the expression vector pFG to generate the pFLG ，in which contained Flk-1 promoter to drive Lmo4 expresses in only FLK-1 + cells．The mESC were transfected with pFG or pFLG plasmids and subsequently screened with geneticin ( G418) to produce cell clones． These cell clones were named mESC /pFG and mESC /pFLG ，respectively． The mESC /pFG and mESC /pFLG were cultured in the differentiation medium for either 4 days or 10 days to generate embryoid bodies (EB) ．The 10-day embryoid bodies ( 10 d-EBs) carrying the pFG and pFLG vectors were subsequently stimulated to generate the blast-colony forming cells (BL-CFC) ，which indicated the presence of hemangioblasts．The endo- thelial cell sprouting analysis was performed by using 10 d-EBs．The expression of the interest genes was detected by using qualitative RT-PCR or Western blot analysis.
Results :The pFLG expression vector was successfully con- structed through PCR identification．The mESC /pFG and mESC /pFLG cells were obtained after transfected with the pFG or pFLG vectors and selected by G418．The cells spontaneously differentiate to generate EBs，in which some green fluoresce cells were present．Western blot analysis showed that a significant increase in LMO4 expression in both 4 d-EB and 10 d-EB when compared to mESC．BL-CFC analysis showed that the 4 d-EB/ pFLG had a higher cloning efficiency ( 7. 70% ± 1. 27% ) ，comparing with that of the 4 d-EB/ pFG ( 1. 15% ± 0. 48% ) ( P = 0. 021) ．Quantitative RT-PCR results showed that the expression of Flk-1，C-kit，Tie-2 and Ve-cad genes in 10 d- EBs /pFLG increased more than 2-fold compared to 10 d-EBs /pFG．The endothelial cell sprouting analysis result showed a significant increase in the number and length of new blood vessels in 10 d-EB/ pFLG compared to 10 d- EB/ pFG (P＜0. 05) ．
Conclusion :Overexpression of LMO4 promotes hemangioblast differentiation from mESC， and benefits for endothelial cell differentiation and angiogenesis.
- Full text:LMO4在小鼠胚胎干细胞分...内皮细胞和血管生成中的作用_项明华1.pdf
