The Study Of Effect And Mechanism Of Picosecond Pulsed Electric Field On Cervical Carcinoma Angiogenesis In Vitro

Pulsed electric field(PEF) is a new kind of minimally invasive and local ablation technology in recent years, which by means of the pulse can not only directly lead to cell swelling rupture, also can cause the occurrence of apoptosis. And the latest research progress of found that when the pulse wave width down to picoseconds, using special constructed capable of firing the ultra wideband impulse radiating antenna non-invasive focused to achieve that picosecond pulse electric field(psPEF). Therefore, picosecond pulse electric field in the biomedical field especially for tumor therapy,which has attracted more and more attention and research.Tumor growth and metastasis are closely related to angiogenesis.When the existing vessels can not meet the growth of tumor, the tumor will produce angiogenesis related factors to form new blood vessels. Tumor angiogenesis involves a complicated course such as the proliferation,migration and lumen formation of endothelial cells. At the same time, the regulation of tumor angiogenesis can be precisely controlled by the balance between angiogenic factors and inhibitory factors. The study found thatvascular endothelial growth factor(VEGF) and hypoxia inducible transcription factor HIF-1α is an important contributing factor of angiogenesis.The previous research shows that we have found the effect of picosecond pulse electric field on the proliferation and apoptosis of cervical cancer He La cells. However, there is no relevant report on the effect of the tumor angiogenesis. In order to investigate the effect of picosecond pulse electric field on tumor and its surrounding blood vessels, this experiment took human umbilical vein endothelial cells and human cervical carcinoma He La cells as the research object. This experiment use prophase experimental parameters, with low, medium and high field strength of picosecond pulse(200kv/cm、400kv/cm、600kv/cm) for processing method,application of CCK-8 assay, Transwell chamber method, scratch test,lumen formation assay, flow cytometry detect picosecond pulse electric field treatment on HUVEC proliferation, migration and tube formation, cell cycle and apoptosis effect; by using real-time PCR and Western blot to investigate the changes of expression levels of mRNA and protein in angiogenesis related factors after picosecond pulse electric field treatment on HeLa cells at the molecular level, and with that We can further explore the possible molecular mechanism of pulsed electric field on the formation of tumor angiogenesis, and to provide scientific basis for the follow-up of animal experiments.Part one Effect of picosecond pulsed electric field on biological behavior of HUVECObjective: To investigate the effect of proliferation, migration, tube formation, cell cycle and apoptosis of picosecond pulsed electric field(psPEF) on human umbilical venous endothelial cells(HUVEC) in vitro.Methods: psPEF with constant parameters(pulse duration of800 ps,recurrence rate of 3Hz,and the number of pulses 2000) and three levels electric field strength(200KV/cm,400 KV/cm and 600 KV/cm) were performed on HUVEC. CCK-8 assay was used to detect the cell proliferation of HUVEC treated with psPEF for 2h, 4h, 6h, 8h, 10 h, 12 h,14h, 16 h. The effect of ps PEF on HUVEC migration was measured by wound healing method and Transwell assay.The formation of vascular lumen in HUVEC cells was observed by the experiment of lumen formation. The apoptosis and cell cycle was detected by flow cytometry.Results: The death rate of HUVEC went up with increasing of the amount of pulses, and the maximum inhibitory rate was 12 hours after the treatment with ps PEF. ps PEF inhibited dose-dependently the migration and vascular lumen formation of HUVEC. After treated with psPEF, the results of the cell cycle indicated that psPEF arrest cell cycle in G2/M phase,and the apoptosis rate was increased.Conclusion:ps PEF inhibits the migration, proliferation and vascular lumen formation of HUVEC, as well as induces apoptosis and arrests the cell cycle in G2 / M phase.Effect of picosecond pulsed electric field on angiogenesis related factors in human cervical cancer He La cellsObjective: To investigate the effect of picosecond pulsed electric field on angiogenesis related factors in human cervical cancer HeLa cells.Methods: HeLa cells were divided into four groups: control group and low, medium and high field strength(200kV/cm, 400kV/cm,600kV/cm). The parameters of picosecond pulse electric field are: fixed pulse width 800 ps, frequency 3Hz, pulse number 2000. After treated by picosecond pulsed electric field, He La cells was continued to cultivate 12 h at 37 degrees C, 5%CO2 incubator. Expression of angiogenesis related factors HIF1α, VEGF were detected from the levels of transcription and translation by Real time-PCR and Western blot.Results: after the effect of picosecond pulse electric field, HIF1α,VEGF expressions were down regulated at both transcriptional and translational levels.Conclusion: Picosecond pulse can inhibit the expression of VEGFand HIF1α in HeLa cells, and the results may be related to the HIF1α-VEGF signaling pathway.