BK_（Ca）对人子宫内膜容受性的影响及其机制研究
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摘要
时至今日,调控胚胎着床和发育的具体机制仍不明确。成功的着床取决于胚泡的侵入能力和子宫内膜的容受性两方面因素。子宫内膜是个非常独特的组织,它在卵巢性激素的作用下在整个月经周期经历着巨大的变化——经由增生期至分泌期建立起内膜容受状态,为胚胎植入做准备。在一个非常短暂的称为种植窗(wOI)时期,子宫内膜细胞在介导母胎间的信号传导方面发挥着及其重要的作用。它们合成和分泌许多分子,为胚胎着床做准备。
离子通道参与了许多细胞功能的实现,如：合成和分泌内源性因子包括性激素、神经递质。尽管在子宫中已经有一些离子通道被研究,但这些研究绝大多数将重点都着眼于离子通道对子宫肌细胞张力的调控作用。是否有离子通道参与了调控子宫内膜细胞的容受性的建立、调节wOI的表达需要被阐明。
大电导Ca2+激活K+通道(Large conductance Ca2+ -activated K+ channel),也称为Slo、BKCa或Maxik通道,广泛地表达于兴奋和非兴奋细胞中,并参与了许多生理过程,如激素的分泌、神经递质的释放、细胞的兴奋、心率和血管反应性以及平滑肌张力的调控等。BKCa不仅能被细胞内Ca2+离子激活,而且还能被跨膜电位去极化激活。对BKCa的研究至今为止主要集中在平滑肌细胞上,然而在其它的生理过程中它们也具有非常重要的作用,如：1)动作电位复极化；2)调控神经元的兴奋性；3)调控神经递质的释放；4)调控肌细胞的收缩和舒张；5)调控分泌细胞分泌各种激素、因子；6)参与先天免疫的建立；7)影响细胞的增殖、凋亡及肿瘤形成；8)影响细胞的容积、形态等。BKCa通道存在于线虫(新杆状线虫),昆虫(果蝇)和哺乳动物等多种物种中。人类的BKCa基因最先从脑组织中克隆出来。已经知道在人体中脑组织、平滑肌、骨骼肌、耳蜗毛细胞、肾上腺皮质、成牙质细胞、胰岛细胞、结肠及肾脏上皮细胞等组织有BKCa的分布,我们前期的研究发现子宫内膜细胞中也有其表达,然而对其是否参与了子宫内膜容受性的建立,我们仍一无所知。
在本研究中我们进一步验证了BKCa在子宫内膜的表达及其分布规律,探讨了它对WOI因子转录表达的影响、对胚胎着床的影响,并对其机理进行了研究。
第一部分BKCa在人子宫内膜组织中的表达、分布
目的：确认BKCa在人子宫内膜细胞上的表达,及在月经周期不同时期的表达变化,分析BKCa在特定不孕人群中的表达差异。
方法：对80例正常月经周期人子宫内膜组织(其中增生期44例,中分泌期36例)采用qRT-PCR法,Western blotting法半定量检测BKCa的表达水平。用免疫组化法检测BKCa在子宫内膜的定位。用全细胞膜片钳检测细胞膜K+电流的变化。
结果：1.在36例中分泌期子宫内膜中BKCa mRNA和蛋白的表达明显高于44例增生期子宫内膜中BKCa mRNA和蛋白的表达(p<0.05)。
2.管性不孕的妇女接受IVF-ET(?)台疗后治疗失败组(failed subgroups,21例)的中分泌期子宫内膜中BKCa mRNA和蛋白的表达明显低于治疗成功组(successful subgroups,15例)的妇女,(p<0.05)。
3.免疫组化显示BKCa主要定位于子宫内膜细胞的腺上皮和腔上皮,间质细胞上有弱表达。
4.全细胞膜片钳检测显示人子宫内膜细胞有电压依赖的电流,而这种电流可以被0.1μM IbTX特异性的阻断
结论：BKCa表达于人子宫内膜上皮细胞中,其表达水平在月经周期不同时期及在接受IVF-ET后不同妊娠结局的患者的子宫内膜细胞中的差异表达可能和子宫内膜容受性的建立有关。
第二部分BKca参与子宫内膜容受性的调控
目的：应用体外粘附模型和在体动物种植模型来研究BKca是否参与对胚胎粘附和着床的调控,并利用研究BKca功能的工具药IbTX和siRNA干扰技术研究了BKCa对子宫内膜容受性标志性因子——WOI因子的调控。
方法：1.以JAr和Ishikawa细胞构建体外粘附模型,通过siRNA干扰子宫内膜细胞中BKCa表达观察滋养细胞球粘附率的变化,评价BKCa在滋养细胞球粘附到子宫内膜细胞中的作用。
2.建立在体动物胚胎种植模型,以靶向BKCaα亚单位的siRNA干扰受孕小鼠子宫内膜BKCa的基因的表达,观察BKCa对胚胎种植的影响。
3.培养原代子宫内膜细胞,以IbTX为工具药阻断细胞中BKca的功能,qRT-PCR和Western blotting检测细胞中种植窗(WOI)因子(LIF、integrinβ3.claudin-4和DKK-1)的变化。
4.培养Ishikawa细胞,以IbTX为工具药阻断细胞中BKCa的功能,或以靶向BKCa的siRNA干扰细胞中BKCa的表达,qRT-PCR和Western blotting检测细胞中种植窗(WOI)因子(LIF、integrinβ3、claudin-4和DKK-1)的变化。
结果：1.体外粘附模型中,与对照组相比,siRNA干扰组的滋养细胞球粘附率显著降低。而如果用siRNA干扰同时加用LIF处理,则粘附率下降趋势被明显削弱,与对照组相比没有显著性差别。而单用LIF组粘附率没有明显改变。
2.在体动物胚胎种植模型显示,靶向BKCaα亚单位的siRNA成功干扰基因表达后,胚胎着床数明显低于对照组。
3.在原代子宫内膜细胞中,当阻断BKCa的功能时,LIF、integrinβ3、claudin-4和DKK-1的表达水平与对照组相比明显下降,(p<0.05)。
4.在Ishikawa细胞中,当阻断BKCa的功能时,LIF、integrinβ3、claudin-4和DKK-1的表达水平与对照组相比明显下降,(p<0.05);siRNA干扰组Ishikawa细胞中LIF、integrinβ3、claudin-4和DKK-1表达的变化趋势和IbTX组中一致。
结论：子宫内膜细胞中BKCa通过调控WOI因子的表达影响子宫内膜的容受性,进而影响胚胎粘附、着床。
第三部分BKCa调控子宫内膜细胞WOI因子的机制研究
目的：初步探讨子宫内膜细胞中BKCa调控WOI因子表达的可能机制。
方法：1.应用显微荧光测定法检测子宫内膜细胞中Ca2+的浓度,观察IbTX处理组和对照组间Ca2+离子浓度的变化,评价BKCa对细胞内Ca2+离子浓度的影响。
2.应用EMSA法测定IbTX处理组与对照组子宫内膜细胞中NF-κB活性变化,评价BKCa对细胞中NF-κB活化的影响。
3.应用Western blotting(?)去检测IbTX处理组与对照组子宫内膜细胞中IκB-α的降解情况,进一步确定BKCa对细胞中NF-κB活化的影响。
结果：1.ATP刺激子宫内膜细胞会引起细胞内Ca2+浓度明显升高。而IbTX对内膜细胞因ATP刺激引起的胞内Ca2+浓度的升高有明显的抑制作用,(p<0.05)。
2.EMSA检测显示IbTX会显著抑制子宫内膜细胞中性激素诱导的NF-κB的活化,(p<0.05)。
3.Western blotting法检测显示在性激素诱导30分钟时,子宫内膜细胞中IκB-α的降解最为明显,而加用IbTX处理细胞会显著削弱性激素诱导的IκB-α的降解。在60分钟时,性激素加或不加IbTX处理组的细胞中IκB-α的降解已恢复到与对照组相同的水平。
结论：BKca参与调控细胞中Ca2+浓度和NF-κB的活化。BKca可能正是通过这两个途径调节WOI因子的表达,影响子宫内膜细胞容受性,进而影响胚胎的着床。
Biomolecular mechanisms that are involved in the embryo implantation and development process are still unknown. This event, like all complex biological mechanisms, depends on the activity of several factors of both embryonic and endometrial origin. Endometrium is a unique tissue that sustains numerous changes during the menstrual cycle under control of the ovarian hormones, estradiol and progesterone. It shifts from a proliferative state to a secretory state to prepare for establishing receptivity for the embryo implantation. During a short period named the window of implantation (WOI), endometrial cells play a prominent role in the signaling between maternal and fetal tissues. They synthesize and secrete many molecules which modulate uterine function for receipting embryo.
Ion channel activity participates in many cellular functions such as synthesis and secretion of endogenous factors including hormones and neurotransmitters. Although some ion channels have been studied in uterus, most of these researches were focused on the roles of ion channels in controlling uterine muscle constriction and dilation. The roles of ion channels in regulation of endometrial WOI factors and embryo implantation are needed to be elucidated. Large-conductance calcium-activated potassium channels (BKCa channels, also known as Kcal.1, KCNMA1, Slol or maxi-K) are important contributors to excitable cells by regulating the duration of calcium action potential trains, shaping the after-hyperpolarization, and curtailing calcium entry cross membrane. channels are activated both by elevated cytosolic Ca2+ levels and by membrane depolarization. They were mainly studied in smooth muscle cells (vascular, airway, uterine, gastrointestinal, and urinary bladder) where they are the key players in setting the contractile tone. However, they also play important roles in other physiological processes such as action potential repolarization; neuronal excitability; neurotransmitter release; hormone secretion; tuning of cochlear hair cells; innate immunity; cell proliferation, apoptosis and oncogenesis. BKCa channels are present in many members of the animal kingdom such as nematodes (Caenorhabditis), insects (Drosophila), and mammals. The human BKCa gene was first cloned from the brain. Although they are discovered widely distributing in many different tissues in human such as brain (cerebellum, habenula, striatum, olfactory bulb, neocortex, granule and pyramidal cells of the hippocampus), skeletal muscle, smooth muscle (vascular, airway, uterine, gastric, bladder), adrenal cortex, cochlear hair cells, odontoblasts, pancreatic islet cells, colonic and kidney epithelium, and in our previous study we found that BKCa expressed in endometriam, it has been still unclear whether BKCa channels contribute endometrial receptivity.
In the present study we confirm that BKCa channels were expressed in human uterine endometrium and the discipline in which BKCa channels were expressed temporospatially. Using Iberiotoxin (IbTX, a specific BKCa blocker) and siRNA targeting for BKCa gene, we examined the role of BKCa channels in the regulation of WOI factors synthesis in human endometrium. We also examined the possible effect of BKCa channels on embryo implantation. At last, we try to demonstrate the possible machenism。
Part One:The expression of BKCa in human endometrium and its significance
Objective:To confirm whether BKCa channels were expressed in human uterine endometrium as we previous found. To analyse the differential expression of BKCa in infertile women due to tubule pathology.
Methods:qRT-PCR and Western blot assay were used to analyse the expression of BKCa channels in human endometrium derived from 80 patients(44 cases, proliferative phase and 36 cases, mid-secretory phase). Immunohistochemical staining was used to locate BKCa in human endometrium. And Patch-Clamping was used to analyze K+ current in human endometrium.
Results:1. qRT-PCR and Western blotting analysis shows the expression of BKCa in human endometrium at the mid-secretory phase was significantly higher than that at the proliferative phase.
2. The levels of BKCa in the failed subgroup were significantly lower than those in the successful subgroup
3. BKCa Immunohistochemical staining was present in both luminal and glandular epithelial cells in human endometrium. Weak staining was also observed in the stromal endometrium.
4. Patch-clamp experiment demonstrated that a voltage dependent current was detected in human endometrial cell. This current could be significantly inhibited by treatment of cells with 0.1μM IbTX.
Conclusion:BKCa expressed in human endometrium. The differential expression at different time point in menstrual cycle and in different infertile patients may associate with establishment of endometrial receptivity.
Part Two:BKca affect endometrial receptivity via altering the production of WOI factors
Objective:To investigate the effect of BKCa on the attachment of the floating blastocyst to the receptive endometrial epithelium. To examine the potential involvement of BKCa channel in EP-induced alteration of WOI factors in human endometrium.
Methods:1. In Vitro attachment model was employed with simulated embryos to analyze the association between BKCa channel and embryos attachment.
2. qRT-PCR and Western blot assay were used to analyse the alteration of WOI factors in human primary endometrial cells treated with or without IbTX.
3. qRT-PCR, ELISA and Western blot assay were used to analyse the alteration of WOI factors in Ishikawa cells treated with or without IbTX. To confirm the results, siRNA targeting for BKCa channel was employed also.
Results:1. Attachment rate analysis demonstrated that knock-down BKCa by siRNA significantly decreased JAr speroids attachment rate (p<0.05). However supplement LIF partially rescued the decrease of attachment rate induced by suppression of BKCa expression; Supplement LIF alone did not increase attachment rate in srambled siRNA group.
2. qRT-PCR assay showed LIF, integrin p3, claudin-4 and DKK-1 gene expression were significantly increased in EP-stimulating cells compared with control, whereas IbTX could attenuate this effect.
3. qRT-PCR, ELISA and western blot analysis showed that EP could induced increase WOI factors, whereas IbTX significantly attenuated this effect induced by EP in Ishikawa cells. LIF, integrinβ3, claudin-4 and DKK-1 mRNA transcription and protein expression were significantly decreased in Ishikawa cells transfected with BKCa siRNA compared with cells transfected with scrambled siRNA (P<0.5 respectively).
Conclusion:BKCa channels may affect embryo implantation via altering the production of WOI factors in human endometrium.
Part Three:The mechanism involved in regulating WOI factors by BKCa in human endometrium
Objective:To investigate the possible machenism of regulating WOI factors by BKca.
Methods:1. Single-cell microfluorimetry was used to monitor agonist-evoked cytosolic Ca2+ transients in real time to investigate the contribution of BKCa to the increase of Ca2+ in cytoplasm in human endometrial cells.
2.EMS A assay was used to examine whether BKca interfered with the nuclear translocation and activation of NF-κB by EMSA.
3. Western blotting was used to detect the level of IKB-a in human endometrial cells to confirm whether the nuclear translocation of NF-κB was happened.
Results:1. Single-cell microfluorimetry assay indicated elevations in cytosolic Ca2+ were evoked by ATP. However, IbTX attenuated evoked increase of Ca2+ in cytoplasm significantly.
2. EMSA assay showed the levels of the NF-κB activation were significantly decreased in the presence of IbTX, Compared with control.
3. Western blotting indicated IKB-awas degraded significantly 30 min after treatment of cells with EP and reappearing at 60 min, however the effect of IκB-degradation at 30 min after treatment by EP was significantly attenuated in the presence of IbTX
Conclusion:BKCa channels may affect WOI factors expression, regulate endometrial receptivity via altering intracellular Ca2+ level and activation of NF-κB.
离子通道参与了许多细胞功能的实现,如：合成和分泌内源性因子包括性激素、神经递质。尽管在子宫中已经有一些离子通道被研究,但这些研究绝大多数将重点都着眼于离子通道对子宫肌细胞张力的调控作用。是否有离子通道参与了调控子宫内膜细胞的容受性的建立、调节wOI的表达需要被阐明。
大电导Ca2+激活K+通道(Large conductance Ca2+ -activated K+ channel),也称为Slo、BKCa或Maxik通道,广泛地表达于兴奋和非兴奋细胞中,并参与了许多生理过程,如激素的分泌、神经递质的释放、细胞的兴奋、心率和血管反应性以及平滑肌张力的调控等。BKCa不仅能被细胞内Ca2+离子激活,而且还能被跨膜电位去极化激活。对BKCa的研究至今为止主要集中在平滑肌细胞上,然而在其它的生理过程中它们也具有非常重要的作用,如：1)动作电位复极化；2)调控神经元的兴奋性；3)调控神经递质的释放；4)调控肌细胞的收缩和舒张；5)调控分泌细胞分泌各种激素、因子；6)参与先天免疫的建立；7)影响细胞的增殖、凋亡及肿瘤形成；8)影响细胞的容积、形态等。BKCa通道存在于线虫(新杆状线虫),昆虫(果蝇)和哺乳动物等多种物种中。人类的BKCa基因最先从脑组织中克隆出来。已经知道在人体中脑组织、平滑肌、骨骼肌、耳蜗毛细胞、肾上腺皮质、成牙质细胞、胰岛细胞、结肠及肾脏上皮细胞等组织有BKCa的分布,我们前期的研究发现子宫内膜细胞中也有其表达,然而对其是否参与了子宫内膜容受性的建立,我们仍一无所知。
在本研究中我们进一步验证了BKCa在子宫内膜的表达及其分布规律,探讨了它对WOI因子转录表达的影响、对胚胎着床的影响,并对其机理进行了研究。
第一部分BKCa在人子宫内膜组织中的表达、分布
目的：确认BKCa在人子宫内膜细胞上的表达,及在月经周期不同时期的表达变化,分析BKCa在特定不孕人群中的表达差异。
方法：对80例正常月经周期人子宫内膜组织(其中增生期44例,中分泌期36例)采用qRT-PCR法,Western blotting法半定量检测BKCa的表达水平。用免疫组化法检测BKCa在子宫内膜的定位。用全细胞膜片钳检测细胞膜K+电流的变化。
结果：1.在36例中分泌期子宫内膜中BKCa mRNA和蛋白的表达明显高于44例增生期子宫内膜中BKCa mRNA和蛋白的表达(p<0.05)。
2.管性不孕的妇女接受IVF-ET(?)台疗后治疗失败组(failed subgroups,21例)的中分泌期子宫内膜中BKCa mRNA和蛋白的表达明显低于治疗成功组(successful subgroups,15例)的妇女,(p<0.05)。
3.免疫组化显示BKCa主要定位于子宫内膜细胞的腺上皮和腔上皮,间质细胞上有弱表达。
4.全细胞膜片钳检测显示人子宫内膜细胞有电压依赖的电流,而这种电流可以被0.1μM IbTX特异性的阻断
结论：BKCa表达于人子宫内膜上皮细胞中,其表达水平在月经周期不同时期及在接受IVF-ET后不同妊娠结局的患者的子宫内膜细胞中的差异表达可能和子宫内膜容受性的建立有关。
第二部分BKca参与子宫内膜容受性的调控
目的：应用体外粘附模型和在体动物种植模型来研究BKca是否参与对胚胎粘附和着床的调控,并利用研究BKca功能的工具药IbTX和siRNA干扰技术研究了BKCa对子宫内膜容受性标志性因子——WOI因子的调控。
方法：1.以JAr和Ishikawa细胞构建体外粘附模型,通过siRNA干扰子宫内膜细胞中BKCa表达观察滋养细胞球粘附率的变化,评价BKCa在滋养细胞球粘附到子宫内膜细胞中的作用。
2.建立在体动物胚胎种植模型,以靶向BKCaα亚单位的siRNA干扰受孕小鼠子宫内膜BKCa的基因的表达,观察BKCa对胚胎种植的影响。
3.培养原代子宫内膜细胞,以IbTX为工具药阻断细胞中BKca的功能,qRT-PCR和Western blotting检测细胞中种植窗(WOI)因子(LIF、integrinβ3.claudin-4和DKK-1)的变化。
4.培养Ishikawa细胞,以IbTX为工具药阻断细胞中BKCa的功能,或以靶向BKCa的siRNA干扰细胞中BKCa的表达,qRT-PCR和Western blotting检测细胞中种植窗(WOI)因子(LIF、integrinβ3、claudin-4和DKK-1)的变化。
结果：1.体外粘附模型中,与对照组相比,siRNA干扰组的滋养细胞球粘附率显著降低。而如果用siRNA干扰同时加用LIF处理,则粘附率下降趋势被明显削弱,与对照组相比没有显著性差别。而单用LIF组粘附率没有明显改变。
2.在体动物胚胎种植模型显示,靶向BKCaα亚单位的siRNA成功干扰基因表达后,胚胎着床数明显低于对照组。
3.在原代子宫内膜细胞中,当阻断BKCa的功能时,LIF、integrinβ3、claudin-4和DKK-1的表达水平与对照组相比明显下降,(p<0.05)。
4.在Ishikawa细胞中,当阻断BKCa的功能时,LIF、integrinβ3、claudin-4和DKK-1的表达水平与对照组相比明显下降,(p<0.05);siRNA干扰组Ishikawa细胞中LIF、integrinβ3、claudin-4和DKK-1表达的变化趋势和IbTX组中一致。
结论：子宫内膜细胞中BKCa通过调控WOI因子的表达影响子宫内膜的容受性,进而影响胚胎粘附、着床。
第三部分BKCa调控子宫内膜细胞WOI因子的机制研究
目的：初步探讨子宫内膜细胞中BKCa调控WOI因子表达的可能机制。
方法：1.应用显微荧光测定法检测子宫内膜细胞中Ca2+的浓度,观察IbTX处理组和对照组间Ca2+离子浓度的变化,评价BKCa对细胞内Ca2+离子浓度的影响。
2.应用EMSA法测定IbTX处理组与对照组子宫内膜细胞中NF-κB活性变化,评价BKCa对细胞中NF-κB活化的影响。
3.应用Western blotting(?)去检测IbTX处理组与对照组子宫内膜细胞中IκB-α的降解情况,进一步确定BKCa对细胞中NF-κB活化的影响。
结果：1.ATP刺激子宫内膜细胞会引起细胞内Ca2+浓度明显升高。而IbTX对内膜细胞因ATP刺激引起的胞内Ca2+浓度的升高有明显的抑制作用,(p<0.05)。
2.EMSA检测显示IbTX会显著抑制子宫内膜细胞中性激素诱导的NF-κB的活化,(p<0.05)。
3.Western blotting法检测显示在性激素诱导30分钟时,子宫内膜细胞中IκB-α的降解最为明显,而加用IbTX处理细胞会显著削弱性激素诱导的IκB-α的降解。在60分钟时,性激素加或不加IbTX处理组的细胞中IκB-α的降解已恢复到与对照组相同的水平。
结论：BKca参与调控细胞中Ca2+浓度和NF-κB的活化。BKca可能正是通过这两个途径调节WOI因子的表达,影响子宫内膜细胞容受性,进而影响胚胎的着床。
Biomolecular mechanisms that are involved in the embryo implantation and development process are still unknown. This event, like all complex biological mechanisms, depends on the activity of several factors of both embryonic and endometrial origin. Endometrium is a unique tissue that sustains numerous changes during the menstrual cycle under control of the ovarian hormones, estradiol and progesterone. It shifts from a proliferative state to a secretory state to prepare for establishing receptivity for the embryo implantation. During a short period named the window of implantation (WOI), endometrial cells play a prominent role in the signaling between maternal and fetal tissues. They synthesize and secrete many molecules which modulate uterine function for receipting embryo.
Ion channel activity participates in many cellular functions such as synthesis and secretion of endogenous factors including hormones and neurotransmitters. Although some ion channels have been studied in uterus, most of these researches were focused on the roles of ion channels in controlling uterine muscle constriction and dilation. The roles of ion channels in regulation of endometrial WOI factors and embryo implantation are needed to be elucidated. Large-conductance calcium-activated potassium channels (BKCa channels, also known as Kcal.1, KCNMA1, Slol or maxi-K) are important contributors to excitable cells by regulating the duration of calcium action potential trains, shaping the after-hyperpolarization, and curtailing calcium entry cross membrane. channels are activated both by elevated cytosolic Ca2+ levels and by membrane depolarization. They were mainly studied in smooth muscle cells (vascular, airway, uterine, gastrointestinal, and urinary bladder) where they are the key players in setting the contractile tone. However, they also play important roles in other physiological processes such as action potential repolarization; neuronal excitability; neurotransmitter release; hormone secretion; tuning of cochlear hair cells; innate immunity; cell proliferation, apoptosis and oncogenesis. BKCa channels are present in many members of the animal kingdom such as nematodes (Caenorhabditis), insects (Drosophila), and mammals. The human BKCa gene was first cloned from the brain. Although they are discovered widely distributing in many different tissues in human such as brain (cerebellum, habenula, striatum, olfactory bulb, neocortex, granule and pyramidal cells of the hippocampus), skeletal muscle, smooth muscle (vascular, airway, uterine, gastric, bladder), adrenal cortex, cochlear hair cells, odontoblasts, pancreatic islet cells, colonic and kidney epithelium, and in our previous study we found that BKCa expressed in endometriam, it has been still unclear whether BKCa channels contribute endometrial receptivity.
In the present study we confirm that BKCa channels were expressed in human uterine endometrium and the discipline in which BKCa channels were expressed temporospatially. Using Iberiotoxin (IbTX, a specific BKCa blocker) and siRNA targeting for BKCa gene, we examined the role of BKCa channels in the regulation of WOI factors synthesis in human endometrium. We also examined the possible effect of BKCa channels on embryo implantation. At last, we try to demonstrate the possible machenism。
Part One:The expression of BKCa in human endometrium and its significance
Objective:To confirm whether BKCa channels were expressed in human uterine endometrium as we previous found. To analyse the differential expression of BKCa in infertile women due to tubule pathology.
Methods:qRT-PCR and Western blot assay were used to analyse the expression of BKCa channels in human endometrium derived from 80 patients(44 cases, proliferative phase and 36 cases, mid-secretory phase). Immunohistochemical staining was used to locate BKCa in human endometrium. And Patch-Clamping was used to analyze K+ current in human endometrium.
Results:1. qRT-PCR and Western blotting analysis shows the expression of BKCa in human endometrium at the mid-secretory phase was significantly higher than that at the proliferative phase.
2. The levels of BKCa in the failed subgroup were significantly lower than those in the successful subgroup
3. BKCa Immunohistochemical staining was present in both luminal and glandular epithelial cells in human endometrium. Weak staining was also observed in the stromal endometrium.
4. Patch-clamp experiment demonstrated that a voltage dependent current was detected in human endometrial cell. This current could be significantly inhibited by treatment of cells with 0.1μM IbTX.
Conclusion:BKCa expressed in human endometrium. The differential expression at different time point in menstrual cycle and in different infertile patients may associate with establishment of endometrial receptivity.
Part Two:BKca affect endometrial receptivity via altering the production of WOI factors
Objective:To investigate the effect of BKCa on the attachment of the floating blastocyst to the receptive endometrial epithelium. To examine the potential involvement of BKCa channel in EP-induced alteration of WOI factors in human endometrium.
Methods:1. In Vitro attachment model was employed with simulated embryos to analyze the association between BKCa channel and embryos attachment.
2. qRT-PCR and Western blot assay were used to analyse the alteration of WOI factors in human primary endometrial cells treated with or without IbTX.
3. qRT-PCR, ELISA and Western blot assay were used to analyse the alteration of WOI factors in Ishikawa cells treated with or without IbTX. To confirm the results, siRNA targeting for BKCa channel was employed also.
Results:1. Attachment rate analysis demonstrated that knock-down BKCa by siRNA significantly decreased JAr speroids attachment rate (p<0.05). However supplement LIF partially rescued the decrease of attachment rate induced by suppression of BKCa expression; Supplement LIF alone did not increase attachment rate in srambled siRNA group.
2. qRT-PCR assay showed LIF, integrin p3, claudin-4 and DKK-1 gene expression were significantly increased in EP-stimulating cells compared with control, whereas IbTX could attenuate this effect.
3. qRT-PCR, ELISA and western blot analysis showed that EP could induced increase WOI factors, whereas IbTX significantly attenuated this effect induced by EP in Ishikawa cells. LIF, integrinβ3, claudin-4 and DKK-1 mRNA transcription and protein expression were significantly decreased in Ishikawa cells transfected with BKCa siRNA compared with cells transfected with scrambled siRNA (P<0.5 respectively).
Conclusion:BKCa channels may affect embryo implantation via altering the production of WOI factors in human endometrium.
Part Three:The mechanism involved in regulating WOI factors by BKCa in human endometrium
Objective:To investigate the possible machenism of regulating WOI factors by BKca.
Methods:1. Single-cell microfluorimetry was used to monitor agonist-evoked cytosolic Ca2+ transients in real time to investigate the contribution of BKCa to the increase of Ca2+ in cytoplasm in human endometrial cells.
2.EMS A assay was used to examine whether BKca interfered with the nuclear translocation and activation of NF-κB by EMSA.
3. Western blotting was used to detect the level of IKB-a in human endometrial cells to confirm whether the nuclear translocation of NF-κB was happened.
Results:1. Single-cell microfluorimetry assay indicated elevations in cytosolic Ca2+ were evoked by ATP. However, IbTX attenuated evoked increase of Ca2+ in cytoplasm significantly.
2. EMSA assay showed the levels of the NF-κB activation were significantly decreased in the presence of IbTX, Compared with control.
3. Western blotting indicated IKB-awas degraded significantly 30 min after treatment of cells with EP and reappearing at 60 min, however the effect of IκB-degradation at 30 min after treatment by EP was significantly attenuated in the presence of IbTX
Conclusion:BKCa channels may affect WOI factors expression, regulate endometrial receptivity via altering intracellular Ca2+ level and activation of NF-κB.
引文
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19. Tanaka, Y., K. Koike, and L. Toro, MaxiK channel roles in blood vessel relaxations induced by endothelium-derived relaxing factors and their molecular mechanisms. J Smooth Muscle Res,2004.40(4-5):p.125-53.
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1. Horcajadas, J.A., et al., Determinants of endometrial receptivity. Ann N Y Acad Sci, 2004.1034:p.166-75.
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5. Carson, D.D., et al., Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod,2002.8(9):p.871-9.
6. Stewart, C.L., et al., Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature,1992.359(6390):p.76-9.
7. Jaggar, J.H., et al., Calcium sparks in smooth muscle. Am J Physiol Cell Physiol, 2000.278(2):p. C235-56.
8. Jin, W., et al., Relationship between large conductance calcium-activated potassium channel and bursting activity. Brain Res,2000.860(1-2):p.21-8.
9. Wang, Z.W., et al., SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction. Neuron,2001. 32(5):p.867-81.
10. Sausbier, M., et al., Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+ -activated K+ channel deficiency. Proc Natl Acad Sci U S A,2004.101(25):p. 9474-8.
11. Jackson, W.F., Potassium channels in the peripheral microcirculation. Microcirculation,2005.12(1):p.113-27.
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