| Objective:Establish a model of H9c2 cardiac hypertrophic cells to explore how zinc levels in cardiac hypertrophic cells and the expression of zinc transporter Zip7 change,and to verify the effects of zinc homeostasis in cardiomyocytes.Methods:The first part Establishe an experimental model of cardiac hypertrophic cells:We selected H9c2 cardiomyocytes from rat embryonic heart tissue as experimental subjects,and randomly divided them into control group and experimental group under the same culture conditions.The experimental group was used angiotensin II(Ang ? 0.2uM)to induce H9c2 cardiomyocytes cultured into cardiac hypertrophic cells for 24 hours.The morphologies of the two groups were compared by inverted microscope.The mRNA levels of myocardial hypertrophy factors ANP?BNP and ?-MHC were detected by real-time quantitative PCR(Q-PCR).The protein level of BNP was detected by Western Blot(WB).The second part Detect the levels of zinc ion and the expressions of zinc transporter Zip7 in H9c2 cardiomyocytes:After the establishment of the experimental model of cardiac hypertrophic cells,the concentrations of zinc ion in H9c2 cardiomyocytes of the control group and the experimental group were detected by inductively coupled plasma atomic emission spectrometry(ICPOES).The mRNA and protein levels of zinc transporter Zip7 in cardiomyocytes were detected by Q-PCR and Western Blot respectively.The third part Verify the maintenance of zinc homeostasis in the myocardial protection:On the basis of the successful construction of the experimental model of cardiac hypertrophic cells,the group experiments were repeated by exogenous zinc ion interventions:control group(Control),control group plus zinc ion chelating agent 10uM(Control+TPEN),experimental group(Ang ?),experimental group plus zinc ion chelating agent 10uM(Ang ?+TPEN),experimental group plus zinc chloride 1uM and zinc ion carrier(Ang ?+Zn1),experimental group plus chlorination Zinc 5uM and zinc ionophore(Ang ?+Zn5),experimental group plus zinc chloride 10 uM and zinc ionophore(Ang ?+Zn10);Q-PCR and Western Blot were used to detect mRNA and protein levels of BNP and the zinc transporter Zip7 of each group.Results: 1.The morphologies of the two groups of cardiomyocytes were compared by inverted microscope.Compared with the control group,the cell body diameter of the experimental group became longer,the surface area of the cells became larger,and there was no gap between the cells.The results of Q-PCR showed that the mRNA levels of ANP,BNP and ?-MHC in the experimental group were higher than those in the control group(P<0.05).The results of Western Blot showed that the protein level of BNP in the experimental group was higher than that in the control group(P<0.05),and the differences were statistically significant.2.The results of ICPOES showed that the concentrations of zinc ions in the myocardial cells of the experimental groups were significantly higher than those of the control groups(P<0.05).The results of Q-PCR and Western Blot showed that the mRNA and protein levels of the zinc transporter Zip7 in the experimental groups were higher than those in the control group,the differences were statistically significant(P<0.05).3.The results of Q-PCR and Western Blot after regrouping by exogenous zinc ion showed that:(1)The expressions of Zip7 mRNA and protein in H9c2 cardiomyocytes of Control+TPEN group were higher than those of Control group(P<0.05),the difference was statistically significant.(2)The expressions of Zip7 mRNA and protein in H9c2 cardiomyocytes of Ang ?+TPEN group were higher than those of Ang ? group(P<0.05),the difference was statistically significant.(3)The expressions of Zip7 mRNA and protein in H9c2 cardiac hypertrophic cells were lower than those of Ang ? group after zinc chloride intervention(P<0.05).With the increase of zinc chloride concentration,the expression level of Zip7 in cardiomyocytes showed a decreasing trend,the difference was statistically significant.(4)The expressions of BNP mRNA and protein in H9c2 cardiomyocytes of Control+TPEN group were higher than those of Control group(P<0.05),the difference was statistically significant.(5)There was no significant difference between the mRNA and protein expression levels of Zip7 in H9c2 cardiomyocytes of Ang ?+TPEN group and Ang ? group;(6)After the intervention of 1 uM zinc chloride,the expression level of BNP in H9c2 cardiac hypertrophic cells was not significantly different from that in Ang ? group.With the increase of zinc chloride,the expression of BNP in cardiomyocytes was lower than that in Ang ? group(P<0.05),showed statistically significant difference.Conclusion: 1.In this experiment,we successfully used Ang ? to induce H9c2 cardiomyocytes to construct cardiac hypertrophy cells at the cellular level.2.The concentration of zinc ion and the expression level of zinc transporter Zip7 in cardiac hypertrophic cells were significantly increased.3.There was a negative correlation between zinc ion levels in cardiomyocytes and the expression level of zinc transporter Zip7.Supplementations of zinc ions can reduce the BNP level of cardiac hypertrophic cells.It is confirmed that maintaining zinc homeostasis has protective effects on cardiomyocytes,but the specific protective effect mechanisms and clinical application values need to be further studied. |
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