The Preliminary Study Of The Effects Of Gadolinium Chloride On Angiogenesis Of HUVEC In Vitro And Vascular Development Of Zebrafish Embryo

Background:Abnormal embryonic vascular development may be closely related to the occurrence of adverse neonatal outcomes such as preterm delivery,low birth weight,birth defects and stillbirth.Previous studies have suggested that rare earth exposure during pregnancy may be related to abnormal embryonic vascular development,but the relationship between them is still unclear.Objective:The purpose of this study is to study the effects of rare earth gadolinium exposure on the biological behavior and molecular biology of HUVEC angiogenesis in vitro and whether it has adverse effects on the embryonic vascular development of zebrafish,starting with angiogenesis,which is one of the most important links in vascular development,so as to provide experimental basis for further clarifying the relationship between rare earth exposure and adverse birth outcome.Methods:1.Taking the rare earth gadolinium element which is the most exposed in clinic as the object,the classical cell model of human umbilical vein endothelial cell（human umbilical vein endothelial cell,HUVEC）was selected to study the effect of rare earth gadolinium chloride on the vascular biological behavior of HUVEC in vitro:HUVEC was treated with different concentrations of Gd Cl₃,blank control group was set up,the cell proliferation of HUVEC was detected by MTT method,the migration ability of HUVEC was detected by scratch test,and the invasion ability of HUVEC was detected by Transwel1 invasion test.The tube-forming ability of HUVEC was detected by tube-forming test.2.The expression of vascular endothelial growth factor（VEGF）,the most important protein related to angiogenesis,was detected as an index to evaluate the effect of Gd Cl₃on angiogenesis of HUVEC:after HUVEC was treated with different concentrations of Gd Cl₃,the expression of HUVEC VEGF in control group,1umol/L and 10umol/L Gd Cl₃was detected by Western blotting test.3.Zebrafish embryos labeled with vascular fluorescence were selected as the model to dynamically observe the effects of rare earth gadolinium chloride on the vascular development of zebrafish embryos:the vascular changes of control group,2mmol/L,4mmol/L and 6mmol/L Gd Cl₃were observed by stereo fluorescence microscope,and the sprouting number and budding rate of inferior intestinal vessels were calculated.Results:1.Effects of different concentrations of rare earth gadolinium chloride on the proliferation,migration,invasion and tube formation of HUVCE:（1）the cell proliferation of HUVEC was detected by MTT method:compared with the blank control group,the absorbance of cells in 0.01,0.1,1,10,100,500 and1000umol/L Gd Cl₃groups was close to that of the blank control group,and there was no significant difference between the two groups（P>0.05）.The results showed that gadolinium chloride at a certain concentration had no obvious inhibitory effect on the proliferation of HUVEC.Combined with the literature reading,the concentration of the next in vitro experiment was selected as the concentration of 1umol/L and10umol/L,which was close to the proliferation rate of the control group in this experiment.（2）the migration ability of HUVEC was detected by scratch test:compared with the control group,the scratches between HUVEC treated with 1umol/L and 10umol/L Gd Cl₃were significantly narrowed（P<0.001）,and the scratch healing rate between1umol/L and 10umol/L Gd Cl₃groups was significantly different（P<0.0001）,indicating that the migration ability of cells increased with the increase of concentration.（3）the invasive ability of HUVEC was detected by cell invasion test:compared with the control group,the number of cell invasion increased in HUVEC treated with1umol/L and 10umol/L Gd Cl₃（P<0.001）,and there was significant difference between 1umol/L and 10umol/L Gd Cl₃group（P<0.0001）,indicating that the invasive ability of cells increased with the increase of concentration.（4）the tube-forming ability of HUVEC:compared with the control group,the number of tubules in HUVEC treated with 1umol/L and 10umol/L Gd Cl₃increased significantly（P<0.001）,but there was no significant difference in the number of HUVEC tubes between 1umol/L and 10umol/L Gd Cl₃groups（P>0.05）.2.The expression of VEGF was detected by Western blotting test:compared with the control group,the VEGF expression of HUVEC in 1umol/L and 10umol/L Gd Cl₃groups was significantly higher than that in control group（P<0.001）,and there was significant difference in VEGF content between 1umol/L and 10umol/L Gd Cl₃groups（P<0.0001）.3.Observation on the vascular development of zebrafish embryos:compared with the blank control group,the proliferation of inferior intestinal vessels of zebrafish embryos was observed in 2mmol/L,4mmol/L and 6mmol/L Gd Cl₃groups,which showed an increase in the number of budding（P<0.001）and budding rate（P<0.001）.There was significant difference in the number and rate of budding of blood vessels between 2mmol/L and 4mmol/L and 6mmol/L Gd Cl₃groups,and between4mmol/L and 6mmol/L Gd Cl₃groups（P<0.05）.Conclusions:Gadolinium chloride can enhance the migration,invasion and tube-forming ability of HUVCE in vitro,promote the expression of VEGF in HUVEC cells and affect its angiogenesis,while Gd Cl₃at a certain concentration can promote the abnormal proliferation of subintestinal blood vessels in zebrafish embryos,suggesting that rare earth Gd Cl₃may be a potential risk to embryonic vascular development.