Effect Of LIF On The Expression Of VEGF In Human Endometrial Stromal Cells In Vitro

Objective:Our study emphasis on evaluating effects of leukemia inhibitory factor (LIF) on the secretion and expression of vascular endothelial growth factor (VEGF) in human endometrial stromal cells(hESCs) from secretory phase in vitro and its effect on angiogenesis in hunman umbilical vein endothelial cells(HUVECs).To explore the mechanism will provide a better understanding of LIF in the process of angiogenesis regulation during embryo implantation. Methods:hESCs from secretory phase and HUVECs were isolated and cultured in vitro. RT-PCR was used to investigate the expression of LIF receptor(LIFR) mRNA. hESCs were treated with different concentrations of LIF(0,0.01,0.1,1.0,10 ng/ml) respectively for 24 hours. Levels of VEGF mRNA in hESCs were determined by semi-quantitative RT-PCR and real-time quantitative PCR. The concentration of LIF which could remarkably enhance the expression of VEGF mRNA was used to stimulate hESCs for 24 hours.The secretion and expression of VEGF in hESCs were determined by Western blot and ELISA. LIF gene was amplified using PCR technique, then inserted into pET28a prokaryotic expression vector. After gene sequencing of the recombinant plasmid, pET28a-LIF was transformed into expressing strain, its protein expression was induced by IPTG, then purified. ICR mice were immunized by the purified protein to obtain antibody. HUVECs were cultured on Matrigel, added the supernatants of the hESCs which treated with LIF or LIF+LIF antibody.Angiogenesis was tested by network formation of HUVECs. To explore the direct effection of LIF on HUVECs, HUVECs were treated with different concentrations of LIF(0,0.01,0.1,1.0,10 ng/ml) respectively for 24 hours. The proliferation of HUVECs were tested by MTT colorimetric assay and VEGF mRNA were determined by real-time quantitative PCR.Results:hESCs from secretory phase and HUVECs were isolated and cultured in vitro successfully. Both the cells expressed LIF receptor(LIFR) mRNA. The mRNA expression of VEGF121 and VEGF165 were detected in any fractions. LIF(0.1,1.0,10 ng/ml) augmented the expression of VEGF121 and VEGF165 mRNA(P<0.05). LIF also increased the secretion and expression of VEGF in hESCs. We have successfully amplified LIF gene by PCR technique, obtained highly LIF expressive strain and the recombinant human LIF protein which molecular weight was about 25 kilodalton(kD).The polyclonal antibody of LIF was prepared by immunizing ICR mice.No networks formed in control while there were tube formations on matrigel in the supernatants of the hESCs culture groups treated by LIF, which could be abolished by LIF polyclonal antibody. MTT and real-time quantitative PCR suggested that LIF could not directly induce the proliferation of HUVECs or induce the expression of VEGF mRNA levels of HUVECs.Conclusion:LIF induces the expression of VEGF in hESCs. This may contribute to the development of the angiogenesis of embryo implantation.