| ã€Subject】Much of our understanding and knowledge of human parturition has been blurred by conjecture and extrapolation.Preterm birth(PTB) is the leading cause of neonatal morbidity and mortality.Despite the existence of strategies for treatment and much research has been performed in an attempt to identify novel approaches to prevent preterm delivery,the frequency of PTB has not changed significantly during the past three decades.The mechanisms underlying the initiation of both preterm and term labor remain largely unknown.But we can observe the first step of them—contraction of human myometrium.Uterine myometrial contractility at term is triggered by a number of physiological signals,which orchestrate changes in uterine excitability via ion-channel modulation.The large-conductance calcium-activated potassium channel(BKCa) is the predominant potassium channel type expressed in nonpregnant and pregnant human myometrium.This channel is unique in that it is activated by membrane depolarization and by an increase in the intracellular calcium concentration,thereby playing a pivotal role in modulating uterine function.The smooth muscle BKca channel is formed by tetrameric assembly of an a-subunit and an accessory b-subunit.The a-subunit is the pore-forming part of the BKCa channel.The b-subunit is a key determinant of BKca-channel Ca2+ sensitivity.Recently,it has been suggested that Corticotropin-Releasing Hormone(CRH) acts as a placental clock controlling the length of human gestation.Maternal plasma CRH is low in the first trimester,rises from midgestation to term,and dramatically increases further during labor.Eevated CRH levels have been associated with premature labour, suggesting that the hormone may be involved in regulating the contraction of myometrium.Like other cAMP-producing hormones in the myometrium such asβ2 agonists,CRH may play a part in maintaining uterine quiescence. During pregnancy,CRH levels of maternal plasm increase progressively.Does CRH have an direct effect on the synthesis of BKCa ion channel proteins? Is it possible that CRH decrease the excitability of the myocyte and exert its uterus quiescence function? These issues remain to be elucidated.This research was designed to investigate the expression of BKca-channel in myometrium and whether CRH had a regulation effect on it.ã€Methods】:1,Isolation and culture of human myometrial cells:All biopsies were taken from the lower isthmic part of uteri.After co-digestion with collagenaseâ…¡and DNAaseâ… .2,Reverse transcription and real-time PCR:Total RNA was extracted from cells after being incubated with CRH of different concentrations(0,10-10 mol/L,10-9 mol/L,10-8mol/L,10-7 mol/L) for 24 hours.Quantification of total RNA was performed by measuring absorbance at OD260.cDNA synthesis from total RNA(2ug) was carried out in a reaction volume of 25ul.Aliquots of cDNA from RT reactions were used in real-time PCR to amplify actin and BKca-channelαsubunit andβsubunit cDNA with specific primers.The ratio of the concentration of BKca-channelαsubunit andβsubunit to that of actin was calculated to determine the expression level of BKca.3,Statistics:Data were expressed as mean+S.E.mRNA and protein abundance were expressed relative to a constitutively expressed gene,actin,for the same sample.All of the data were tested for normal distribution and homogeneity of variance with SPSS11.0. One-way ANOVA were used to analyze these data if they were normally distributed and with homogeneity of variance.Otherwise,nonparametric tests of significance were used. All tests were two sides and significance was accepted at a P value＜0.05.ã€Results】1,Myometrial cells acquired by enzymatic digestion with collagenase or tissue culture displayed typical morphous of long slender fusiform.2,The effect of CRH and Antalarmin or astressin 2B on BKca channelαsubunit mRNA expression in TNL group.In TNL group,compared with the blank control group,every treaments up-regulated the expression levels of BKca-channelαsubunit mRNA(p=0.047).The effects of CRH of 10-8 mol/L and 10-7mol/L were significantly different from that of control group(p=0.015;0.003).The effects of CRH of 1010 mol/L and 10-9mol/L were not significantly different from that of control group(p=0.037;0.266).Compared with the blank group,Antalarmin or astressin 2B(10-7 mol/L ) alone or together with CRH all up-regulated BKca-channel a subunit mRNA expression level of myometrial cells,but the difference is not significant(p＞0.05).3,The effect of CRH on BKca channelαsubunit mRNA expression in TL group.In TL group,compared with the blank control group,every treaments up-regulated the expression levels of BKca-channelαsubunit mRNA,but the difference is not significant(p =0.904).Compared with the blank group,Astressin(10-8 mol/L) alone or together with CRH all up-regulated BKca-channel a subunit mRNA expression level of myometrial cells,but the difference is not significant(p＞0.05).4,The effect of CRH on BKca channelβsubunit mRNA expression in TNL group.In TNL group,compared with the blank control group,the effect of CRH on BKca channelβsubunit mRNA expression is not clear,the difference is not significant(p= 0.940).Compared with the blank group,Antalarmin or astressin 2B(10-7 mol/L ) alone or together with CRH all up-regulated BKca-channelβsubunit mRNA expression level of myometrial cells,but the difference is not significant(p＞0.05).5,The effect of CRH on BKca channelβsubunit mRNA expression in TL group.In TL group,compared with the blank control group,CRH down-regulated the expression levels of BKca-channelβsubunit mRNA(p=0.027).The effects of CRH of 10-8 mol/L and 10-7 mol/L were significantly different from that of control group(p= 0.045;0.032).The effects of CRH of 10-10 mol/L and 10-9mol/L were not significantly different from that of control group(p=0.781;0.864).Compared with the blank group,Astressin(10-8 mol/L) alone or together with CRH all up-regulated BKca-channelβsubunit mRNA expression level of myometrial cells,but the difference is not significant(p＞0.05).ã€Conclusions】1.CRH of physiological concentrations(10-8 mol/L to 10-7 mol/L) could up-regulate the expression of BKca channel mRNA.2.In TNL group,CRH up-regulated the expression levels of BKca-channelαsubunit mRNA which may maintain uterine quiescence;In TL group,CRH down-regulated the expression levels of BKca-channelβsubunit mRNA which may may be involved in regulating the contraction of myometrium.
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