Study Of 1α, 25-dihydroxyvitamin D₃ On The Regulation Of Calbindin-D28k Expression In Human Tubal Epithelial Cells

Objectives:1.To investigate the expressions and changes of vitamin D receptor(VDR) protein and mRNA during the different menstrual stages in the mucosal epithelium of human fallopian tubes,and clarify whether the VDR expression would exist in human fallopian tubes.2.To detect the expression and change of VDR protein in the human tubal mucosal epithelium of tubal pregnancy,and analyze the potential relationship between the VDR expression and the occurrence of tubal pregnancy.3.To culture human tubal epithelial cells in vitro,and observe the effect of 1α,25-dihydroxyvitamin D₃(1α,25-(OH)₂D₃) on proliferation of human tubal epithelial cells in vitro culture.4.To detect the effects of 1α,25-(OH)₂D₃ on the expressions of VDR,calbindin-D28k (CaBP-D28k) mRNA and protein in human tubal epithelial cells in vitro culture,and analyze the potential regulative roles of 1α,25-(OH)₂D₃ on the VDR,CaBP-D28k expressions in human tubal epithelial cells,furthermore,study the potential regulatory mechanisms of 1α,25-(OH)₂D₃ on the CaBP-D28k expression in human tubal epithelial cells.Methods:1.The expressions of VDR protein and mRNA in the mucosal epithelium of human fallopian tubes were determined by immunohistochemistry(IHC) and reverse transcription polymerase chain reaction(RT-PCR).2.The expression of VDR protein in the human tubal mucosal epithelium in both implantation and nonimplantation sites of tubal pregnancy was detected by IHC.3.The human tubal epithelial cells mechanically isolated,purified by differential time adherent method were cultured in vitro.The epithelial cells' purity was identified by cytokeratin immunocytochemical staining.A culture system of human fallopian tube epithelial cells was established.The effect of 1α,25-(OH)₂D₃ on proliferation of human tubal epithelial cells in vitro culture was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT).4.The effects of 1α,25-(OH)₂D₃ on the expressions ofVDR,CaBP-D28k rnRNA and protein in human tubal epithelial cells in vitro culture were detected by RT-PCR and Western blot (WB).Results:1.The expressions of VDR protein and mRNA in the mucosal epithelium of human fallopian tubesThe expressions of VDR protein and mRNA were detected in the mucosal epithelium of human fallopian tubes.The VDR positive staining was mainly located in the nucleus of the epithelial cells,observed in some smooth muscle cells occasionally.There was no significant difference in the VDR positive expression among the isthmic,ampullary and fimbrial portions during the same phase of menstrual cycle(P＞0.05).However,the VDR positive expression in the different portions changed with the variation of menstrual cycle.It was in a low level during both the early-proliferative and mid- and late-secretory stages,then increased in both the mid- and late-proliferative and early-secretory stages significantly(P＜0.01),and achieved the maximum during the early-secretory stage.The expression of VDR mRNA in the ampullary portion of the human tubal epithelium was in a low level during both the early-proliferative and mid- and late-secretory stages,then obviously increased in both the mid- and late-proliferative and early-secretory stages(P＜0.01).2.The expression of VDR protein in the human tubal mucosal epithelium of tubal pregnancy The expression of VDR protein was detected in the human tubal mucosal epithelium in both implantation and nonimplantation sites of tubal pregnancy.The VDR positive staining was mainly located in the nucleus of the epithelial cells,observed in some smooth muscle cells occasionally.The VDR positive expression of the human tubal mucosal epithelium was in a low level in implantation site,however,increased in nonimplantation site significantly (P=0.017,P＜0.05).3.The culture and identification of human tubal epithelial cells and the effect of 1α,25-(OH)₂D₃ on proliferation of the cells in vitro cultureThe human tubal epithelial cells mechanically isolated,purified by differential time adherent method could be cultured and subcultured in vitro.There was no difference in the growth status of the epithelial cells between the follicular and luteal phase in vitro.From the primary culture cells to the fourth generation cells,the percentage of cytokeratin positive staining cells to the total culture cells exceeded 90%,which could be used in the experiments below.After the different time of the different 1α,25-(OH)₂D₃ concentration treatments,the proliferation(A₄₉₀) of human tubal epithelial cells did not change(P＞0.05).4.The effects of 1α,25-(OH)₂D₃ on the expressions of VDR,CaBP-D28k mRNA and protein in human tubal epithelial cells in vitro cultureAfter 12 h of the different treatments,compared with the blank and alcohol control groups,the expression levels of VDR and CaBP-D28k mRNA in the 10^-7 mol/L 1α,25-(OH)₂D₃ treatment group were increased remarkably(P＜0.05 or 0.01).And there was a positive correlation for the the mRNA expression level between VDR and CaBP-D28k in cells of the 10^-8 mol/L 1α,25-(OH)₂D₃ treatment group(r = 0.968,P=0.032,P＜0.05).After the different time of the 10^-7 mol/L 1α,25-(OH)₂D₃ treatment,compared with control(0 h), the expression levels of VDR,CaBP-D28k mRNA in the 6 h,12 h treatment groups,the expression levels of VDR,CaBP-D28k protein in the 24 h,48 h treatment groups were significantly higher(P＜0.05 or 0.01),respectively.And there were positive correlations between VDR and CaBP-D28k for the rnRNA expression level in 0 h,3 h,6 h,24 h,48 h treatment groups(r = 0.968,0.977,0.973,0.976,0.993),for the protein expression level in each treatment group(r = 0.981,0.982,0.962,0.984,0.985,0.977),respectively(P＜0.05 or 0.01). Conclusions:1.The expressions of VDR protein and mRNA would exist in the mucosal epithelium of human fallopian tubes.And the expression levels would exhibit a cyclic change.During both the mid- and late-proliferative and early-secretory stages,the expression levels of VDR protein and mRNA would increase significantly.2.The expression of VDR protein in the human tubal mucosal epithelium would be different between implantation and nonimplantation site of tubal pregnancy.Compared with implantation site,the expression level of VDR protein in nonimplantation site would increase significantly.3.The human tubal epithelial cells mechanically isolated,purified by differential time adherent method could be cultured with a higher purity in vitro.1α,25-(OH)₂D₃ would not affect proliferation of human tubal epithelial cells in vitro culture.4.1α,25-(OH)₂D₃ might up-regulate the expression levels of CaBP-D28k mRNA and protein through VDR pathway in human tubal epithelial cells in vitro culture.