The Effect Of PBX1 In Human Umbilical Cord Mesenchymal Stem Cells Induced By Methylmercury And Its Related Mechanism

Background:Methylmercury(MeHg)is hundreds of times more toxic than mercury.After entering the body,it can be found in all tissues and organs of the body.It also has fat soluble properties to pass through the placental barrier and can be toxic to the developing fetus.The umbilical cord is the link that connects the mother and the fetus,and contains a large number of umbilical cord mesenchymal stem cells.The MeHg content in the umbilical cord tissue can be used as a biomarker of fetal exposure to methylmercury.Therefore,MeHg may damage the umbilical cord mesenchymal stem cells in the umbilical cord tissue,affect the growth and development of the fetus,and threaten the health of pregnant women and developing fetuses.PBX1 is a member of the homeobox gene family and it has the function of promoting the proliferation of a variety of cells,participates in important processes such as embryonic development and tissue differentiation,has the specificity and affinity for binding to DNA.In this study,the hUCMSCs injury model was established by MeHg exposure,and the cell model was established by overexpression of PBX1,so as to fully understand the toxic effect of MeHg on hUCMSCs,and preliminarily explore the protective effect and mechanism of PBX1 in MeHg induced hUCMSCs injury.It provides new ideas for the study of toxicology experiments at the stem cell level,and provides a theoretical basis for hUCMSCs damage repair research.Aim:To study the toxic effect of MeHg on hUCMSCs,and to explore the effect and mechanism of PBX1 on MeHg induced hUCMSCs damage.Method:1.After hUCMSCs were given a certain dose of MeHg,cell survival rate was detected by MTT method,cell morphological changes were observed under microscope,and apoptosis rate was detected by Annexin V/PI method.Western Blot method was used to detect the expression of DNA damage related proteins(γH2AX,PARP1,RAD51,KU70,KU80)and apoptosis related proteins(PARP1,PAR,AIF,Cleaved Caspase 3,Cyt C)mediated by the mitochondrial pathway.2.Overexpression of PBX1 by lentivirus packaging method,flow cytometry to detect cell apoptosis rate,Western Blot to detect DNA damage related proteins(γH2AX,PARP1,RAD51,KU70,KU80)and mitochondrial pathway mediated apoptosis related proteins(PARP-1,PAR,AIF,Cleaved Caspase 3,Cyt C)expression levels,preliminary study whether PBX1 has a protective effect on MeHg induced cell damage and its mechanism.Result:1.Injury effect of MeHg on hUCMSCs(1)Observe the cell morphology changes of hUCMSCs treated with different concentrations of MeHg for 24 h under the microscope.With the continuous increase of MeHg concentration,the cell refractive index changes,the cell volume becomes larger and then shrinks,rounds,or even falls off,and the number of viable cells decreases.(2)MTT method was used to detect the cell viability of different concentrations(0,2.5,5,7.5,10,12.5 μM)MeHg treatment for 24 h and 7.5 μM MeHg treatment for different time.As the concentration of MeHg treatment increases and the duration of action increases,the cell survival rate decreases.(3)After treatment with different concentrations of MeHg for 24 h,as the concentration increased,Annexin V/PI results showed that the number of Annexin V and PI positive cells increased significantly.The results of flow cytometry showed that the rate of cell apoptosis was significantly increased.(4)Western Blot results showed that as the concentration increased,the expression of PARP-1(116 kDa)and AIF 67 kDa protein decreased.PAR protein and AIF 57 kDa protein showed an upward trend.The protein expression of Cyt C and Cleaved Caspase 3 increased.As time increased,the protein expression of γH2AX gradually increased,PARP-1(116 kDa),RAD51,KU70,and KU80 decreased.2.The protective effect and mechanism of PBX1 on MeHg induced damage to hUCMSCs(1)The apoptotic rate of the PBX1 group was lower than the Control group and the Vector group;the apoptotic rate of the PBX1+MeHg group was lower than the MeHg group.(2)The expressions of PARP1(116 kDa),PAR,AIF(57 kDa),Cleaved Caspase3,and Cyt C in the PBX1+MeHg group were lower than those in the MeHg group and the Vector+MeHg group.(3)The protein expressions of PARP1(116 kDa),KU80,KU70,RAD51,andγH2AX in the PBX1+MeHg group were lower than those in the MeHg group and the Vector+MeHg group.Conclusion:1.Methylmercury can induce hUCMSCs DNA damage and apoptosis mediated by mitochondrial pathway.2.PBX1 can suppress the DNA damage and mitochondrial pathway mediated apoptosis of hUCMSCs caused by methylmercury.