RESUMEN
Objective To investigate the effect and mechanism of estrodial (E2) on intracellular free calcium in the endometrial-myometrial interface (EMI) smooth muscle cells from uteri with adenomyosis.MethodsFrom March 2011 to October 2011,16 uterus specimens were collected from patients with adenomyosis undergoing hysterectomy in Beijing Obstetrics and Gynecology Hospital,which included 9 proliferative endometrium and 7 secretory endometrium.EMI smooth muscle cells from the uterus were cultured and loaded with calcium ion ( Ca2 + ) fluorescent probe fluo-4/AM.The labeled cells were stimulated with the various concentration of E2 ( 1 × 102,1 × 103,1 × 104,1 × 105 pmol/L,respectively),then the changes of intracellular Ca2+ fluorescence intensity were measured by laser scanning microscopy.The most suitable concentration of E2 was selected,and the reaction difference between the EMI smooth muscle cells of two menstrual phases were also investigated; The changes of intracellular Ca2 + fluorescence intensity were detected proliferative and secretory smooth muscle cells in E2 conjugated to bovine serum albumin (17β-E2-BSA) group,cycloheximide (CHX) group,fulvestrant (ICI182780) group and pertussis toxin (PTX) group.Results ( 1 ) The cell viability of primary cultured EMI smooth muscle cells was well at 24 hours culture.(2) 1 × 102 - 1 × 105 pmol/L E2 can rapidly increase the intracellular Ca2+ fluorescence intensity within 1 min ( P ＜ 0.01 ) ;The increased amplitudes caused by 1 × 104 pmol/L and 1 × 105 pmoL/L E2 were the most significant,but there was no significant difference between them (P ＞0.05).1 × 104 pmol/L was the most suitable concentration.( 3 ) With the 1 × 104 pmol/L E2,the Ca2+ fluorescence intensity changes showed no significant difference between the EMI smooth muscle cells from the proliferative phase and secretory phase uterus (P ＞ 0.05 ).The Ca2+ fluorescence intensity changes were 646 ± 32 in 17β-E2-BSA group and 602 ±31 in CHX group,when compared with 513 ±26 and 617 ±35 in respective control group,no significant difference was observed (P ＞ 0.05 ).The increased amplitude of 188 ± 20 in the PTX group and 302 ± 11 in ICI182780 group exhibited significant difference with 632 ± 33 and 635 ± 24 in respective control group ( P ＜ 0.01 ).Conclusion E2 could increase the intracellular Ca2 + of EMI through a membrane receptor dependent and nongenomic mechanism of action.