Title17β雌二醇对子宫腺肌病患者子宫内膜-肌层交界区平滑肌细胞内游离Ca2+浓度的调节作用及机制研究
Other TitlesMechanism of 17β-estrogen on intracellular free calcium regulation in smooth muscle cells at the ;endometrial-myometrial interface in uteri with adenomyosis
Authors汪沙
段华
张颖
王丽平
张恒辉
李国力
Affiliation100006首都医科大学附属北京妇产医院妇科微创中心
北京大学人民医院肝病研究所
100006首都医科大学附属北京地坛医院传染病研究所
Keywords子宫腺肌病
雌二醇
子宫内膜
子宫肌层
肌细胞,平滑肌
缝隙接合部

Adenomyosis
Estradiol
Endometrium
Myometrium
Myocytes,smooth muscle
Gap junctions
Calcium
Issue Date2015
Publisher中华妇产科杂志
Citation中华妇产科杂志.2015,(7),510-515.
Abstract目的:探讨17β雌二醇(17β-E2)对子宫腺肌病患者子宫内膜-肌层交界区(EMI)平滑肌细胞内游离Ca2+浓度([Ca2+]i)的调节作用及可能机制。方法选择2011年9月至2012年12月在首都医科大学附属北京妇产医院妇科微创中心因子宫腺肌病行子宫全切除术的患者28例为腺肌病组,选择同期因子宫颈上皮内瘤变(CIN)Ⅲ行子宫全切除术者31例为对照组,对两组患者的EMI平滑肌细胞进行分离及原代培养。应用Ca2+荧光探针Fluo-4/AM负载平滑肌细胞,采用细胞外钙阻断实验、细胞内钙释放阻断实验、细胞膜钙通道阻断实验、细胞膜钙激活钾通道阻断实验阻断引起[Ca2+]i变化的各个环节,利用激光共聚焦显微镜观察17β-E2诱导后的两组患者EMI平滑肌细胞内[Ca2+]i的变化,其变化程度以ΔF[Ca2+]i表示。结果(1)在常规有Ca2+培养基中,17β-E2诱导后腺肌病组和对照组EMI平滑肌细胞内Ca2+荧光强度均明显高于其17β-E2诱导前,腺肌病组ΔF[Ca2+]i为384±26,对照组ΔF[Ca2+]i为235±20,两组比较,差异有统计学意义(P=0.001);细胞外钙阻断实验显示,细胞外无Ca2+时,腺肌病组ΔF[Ca2+]i为207±17,对照组ΔF[Ca2+]i为221±19,两组比较,差异无统计学意义(P=0.731)。腺肌病组ΔF[Ca2+]i在细胞外有Ca2+时明显高于无Ca2+时,差异有统计学意义(P=0.000);而对照组两者比较,差异无统计学意义(P=0.060)。(2)细胞内钙释放阻断实验显示,阻断肌质网钙库上三磷酸肌醇(IP3)敏感的钙释放通道、肌质网利阿诺定(Ryanodine)受体(RyR)钙释放通道、肌质网钙泵释放后,腺肌病组和对照组ΔF[Ca2+]i分别与其阻断前相比均明显降低(P<0.05),且腺肌病组ΔF[Ca2+]i均明显高于对照组(P<0.05)。(3)细胞膜钙通道阻断实验显示,阻断L-型钙通道后,腺肌病组ΔF[Ca2+]i为211±19,对照组为203±16,两组比较,差异无统计学意义(P=0.064);腺肌病组ΔF[Ca2+]i明显低于其阻断前(ΔF[Ca2+]i为384±28;P=0.001);而对照组与其处理前(ΔF[Ca2+]i为234±22)比较,差异则无统计学意义(P=0.141)。(4)细胞膜钙激活钾通道阻断实验显示,阻断细胞膜钙激活钾通道后,腺肌病组ΔF[Ca2+]i为357±24,对照组为209±19,两组比较,差异有统计学意义(P=0.000);腺肌病组和对照组ΔF[Ca2+]i分别与其处理前(腺肌病组ΔF[Ca2+]i为363±21,对照组为237±20)比较,差异均无统计学意义(P>0.05)。结论17β-E2通过内源性钙库释放Ca2+和细胞膜L-型钙通道介导的Ca2+内流导致子宫腺肌病患者EMI平滑肌细胞内游离[Ca2+]i异常增高,[Ca2+]i调节异常导致的子宫收缩功能障碍可能参与子宫腺肌病的发生、发展。
Objective To investigate the regulation mechanism of estrogen on the free calcium of smooth muscle cells at the endometrial-myometrial interface (EMI) in uteri with adenomyosis. Methods From September 2011 to November 2012, 59 uterine myometrial specimens were obtained from 59 cases underwent hysterectomy, including 28 adenomyosis patients as adenomyosis (ADS) group and 31 patients with cervical intraepithelial neoplasia Ⅲ as control group. EMI smooth muscle cells were cultured and loaded with calcium ion fluorescent probe fluo-4/AM. After treated with trisphosphate (IP3) receptor antagonist, blocker of sarcoplasmic reticulum calcium-adenosine triphosphate (ATP), depleted agent of the ryanodine receptor-operated Ca2+, inhibitor of L-type calcium channel, inhibitor of Na+-Ca2+exchanger, the labeled cells were stimulated with estrogen. The changes of intracellular Ca2+fluorescence intensity were detected by laser scanning microscopy. The changes of intracellular Ca2+concentration was indicated byΔF[Ca2+]i. Results (1) Under normal calcium conditions, after the stimulation of estrogen, intracellular Ca2+fluorescence intensity in ADS group and control group both increased than those without estrogen. TheΔF[Ca2+]i in ADS group was 384±26, and in the control groupΔF[Ca2+]i was 235±20. TheΔF[Ca2+]i in ADS group was higher than that in the control (P<0.01). Without calcium conditions, theΔF[Ca2+]i in ADS group was 207 ± 17, and in the control group ΔF[Ca2+]i was 221 ± 19. TheΔF[Ca2+]i in ADS group was almost the same with the increase in the control (P=0.731). The ΔF[Ca2+]i in ADS group was significantly decreased compared with the calcium condition (P<0.01). However, there was no significant difference in the control between with and without calcium conditions (P=0.060). (2) After treated with IP3 receptor antagonist, blocker of sarcoplasmic reticulum calcium-ATP, depleted agent of the ryanodine receptor-operated Ca2+, theΔF[Ca2+]i in both groups were significantly reduced (P<0.05), the increase in ADS group was significantly higher than that in the control (P<0.05). (3)After treated with inhibitor of L-type calcium channel, theΔF[Ca2+]i in ADS group was 211 ± 19, while in the control group ΔF[Ca2+]i was 203 ± 16, and there was no significantly increased intracellular Ca2 +in both groups (P>0.05). But, the ΔF[Ca2 +]i in ADS group was significantly reduced after treatment compared to before treatment, (211 ± 19 vs 384 ± 28; P=0.001). The increase in control group was almost the same with before (203±16 vs 234±22, P=0.141). (4) After treated with inhibitor of Na+-Ca2+exchanger, theΔF[Ca2+]i in ADS group was 357 ± 24 and in the controlΔF[Ca2+]i was 209±19. The increase in ADS group was significant higher than that in the control (P=0.000). Compared withΔF[Ca2+]i on the condition without treating with inhibitor of Na+-Ca2+exchanger,ΔF[Ca2+]i was 363±21 in ADS group andΔF[Ca2+]i was 237±20 in control group after treatment. When compared with before treatment, there was no significant difference in both groups (P>0.05). Conclusions The increase of intracellular Ca2+induced by estrogen at EMI smooth muscle cells in adenomyosis patients was mostly from the release of arcoplasmic reticulum, and also from the Ca2+influx controlled by L-type calcium channel. The increase of Ca2+inducing abnormal contraction of EMI muscle may have relationship with the development of adenomyosis.
URIhttp://hdl.handle.net/20.500.11897/414597
ISSN0529-567X
DOI10.3760/cma.j.issn.0529-567x.2015.07.007
Indexed中文核心期刊要目总览(PKU)
中国科技核心期刊(ISTIC)
中国科学引文数据库(CSCD)
Appears in Collections:人民医院

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