Correlation Study Of TRPC6 Protein Expression In The Pathogenesis Of Preeclampsia

Part I: Effects of chemical hypoxia on the expression of TRPC6 in trophoblast cellsObjective: To study the application of chemical reagent two cobalt chloride(Co Cl2)to construct the chemical hypoxia model,extravillous trophoblast cells(used in this study is JEG3)in transient receptor cation channel protein 6(transient receptor potential channel 6,TRPC6)expression changes,and changes in intracellular calcium homeostasis,the expression of TRPC6 to investigate the cell after hypoxia and its role in the pathogenesis of preeclampsia.Methods: 1.Extravillous trophoblast cell line JEG3 2.By selecting two chemicals of cobalt chloride(Co Cl2)to establish JEG3 cells under hypoxia model,cells were divided into 3 groups,group with different hypoxia time,divided into control group,hypoxia group and hypoxia 24 h 48h.3.By fluorescence quantitative real-time PCR to detect the content of m RNA was TRPC6,the expression of TRPC6 protein were westen-blot detection method,using fluo-3 to detect the concentration of intracellular calcium.Results: 1.Hypoxia in the presence of TRPC6 increased the expression of m RNA,and in the 24 and 48 hour hypoxia were significantly increased.At the same time,compared with the control,exposure to hypoxia in JEG3 cells,the expression of the TRPC6 m RNA increased about 9 times of.RT-PCR revealed TRPC6 m RNA between hypoxia group and control group the same batch of cells,there were significant differences(P <0.005).2.24 h in hypoxia and 48 h,using the Western Blot method,detecting the expression of TRPC6 in JEG3 cells,We found that 24 h and 48 h in hypoxia,the expression level of TRPC6 protein were increased,and compared with the control group were significantly different(P and <0.005).The expression level of TRPC6 protein in the hypoxia increased with time in a stable level of.3.In this study,we used fluorescent probe Fluo-3 / AM to mark cell calcium the use of Pro-plus image analysis software to analyze the fluorescence image,with the average optical density(IOD SUM /area SUM)to represent the calcium ion concentration in each group.The results show that the normal.The pictures showed similar low fluorescence intensity,while the Fluo-3 / AM fluorescence intensity of the hypoxic cells increased,indicating that high expression of TRPC6 might lead to changes in intracellular free calciumConclusion: The application of two cobalt chloride chemical hypoxia,JEG3 cells increased TRPC6 expression and intracellular calcium concentration also increased.We established the cell model of high expression of hypoxia induced TRPC6 JEG3 cell line,found that hypoxia can induce high expression of TRPC6 and hypoxia itself is an important cause of preeclampsia early onset,therefore we can think of the cell model are similar in patients with preeclampsia trophoblast cells,using it we can further explain the high expression of TRPC6.Part II:TRPC6 protein affects extravillous trophoblasts JEG3 biology behavior in the pathogenesis of preeclampsiaMethods: 1.Using lentiviral packaging containing rs3824934 sequence of TRPC6 protein overexpression intervention under normal culture of extravillous trophoblast cells were observed under fluorescence microscope JEG3.2.Lentivirus transfection,the transfection efficiency was detected more than 80%,and the expression of TRPC6 protein in extravillous trophoblast cells cultured JEG3 PCR were detected by fluorescence quantitative.3.In the TRPC6 to carry,once again verified whether the experiment is successful.To determine the expression of CASP PCR detected apoptosis factor TRPC6 protein expression in the cells by fluorescence quantitative The expression of ase3,12,then the method of using Western blot to detect the expression of different groups of cells.Application of CCK8 detection method to detect trophoblastic proliferation changes.Results: 1.Will carry protein transfected with TRPC6 overexpression were blank group and negative control and the negative control group and TRPC6 overexpression results for statistical analysis,we found no significant difference between the groups(t=2.061,P <0.05),and negative control group and TRPC6 overexpression group have significant difference(t=9.390,P <0.05),lentivirus transfected successfully.2.After overexpression of TRPC6,compared with the negative control group,the expression of apoptosis factor Caspase3 and caspase12 m RNA also increased,the former increased About 32 times,which increased about 8 times.RT-PCR experiments revealed the same batch of cells in the TRPC6 expression between Caspase3 group and negative control group(t=14.97,P <0.05)and caspase12(t=6.827,P <0.05)the expression of.3.Had significant difference in TRPC6 expression of stable strain after use Western Blot were detected in two groups of cells Caspase3 and the expression of caspase12 protein,found over expression in group Caspase3(t =13.08,P <0.05)and caspase12(t=15.45,P <0.05)protein expression levels were increased,and compared with the negative control group were significantly different.4.CCK8 cell proliferation assay showed that: compared with the negative control group,TRPC6 overexpression group of trophoblast cells od were significantly decreased,the difference was statistically significant(t=5.378,P<0.05).And we also found that trophoblast cells proliferation associated with culture time,culture of 24 h,the OD value of trophoblast cell proliferation is the strongest,highest,followed with With the extension of the culture time,OD value of trophoblast cells decreased,the proliferation gradually decreased,the difference was statistically significant(one-way,ANOVA,F=40.76,P <0.05).Conclusion: TRPC6 protein can significantly reduce the ability of trophoblast proliferation,so we can infer that TRPC6 is affected by the ability of proliferation and apoptosis of trophoblast cells,resulting in shallow placental implantation,placental vascular remodeling effects,and thus participate in preeclampsia occurrence.