Wang Sha, Duan Hua, Zhang Ying, Wang Liping, Zhang Henghui, Li Guoli
Department of Gynecology Minimally Invasive Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China.
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Zhonghua Fu Chan Ke Za Zhi. 2015 Jul;50(7):510-5.
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.
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 Ca(2+), inhibitor of L-type calcium channel, inhibitor of Na(+)-Ca(2+) exchanger, the labeled cells were stimulated with estrogen. The changes of intracellular Ca(2+) fluorescence intensity were detected by laser scanning microscopy. The changes of intracellular Ca(2+) concentration was indicated by △FCa(2+).
(1) Under normal calcium conditions, after the stimulation of estrogen, intracellular Ca(2+) fluorescence intensity in ADS group and control group both increased than those without estrogen. The △FCa(2+) in ADS group was 384 ± 26, and in the control group △FCa(2+) was 235 ± 20. The △FCa(2+) in ADS group was higher than that in the control (P < 0.01). Without calcium conditions, the △FCa(2+) in ADS group was 207 ± 17, and in the control group △FCa(2+) was 221 ± 19. The △FCa(2+) in ADS group was almost the same with the increase in the control (P = 0.731). The △FCa(2+) 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 Ca(2+), the △F[Ca(2+)]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, the △FCa(2+) in ADS group was 211 ± 19, while in the control group △FCa(2+) was 203 ± 16, and there was no significantly increased intracellular Ca(2+) in both groups (P > 0.05). But, the △FCa(2+) 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(+)-Ca(2+) exchanger, the △FCa(2+) in ADS group was 357 ± 24 and in the control △FCa(2+)was 209 ± 19. The increase in ADS group was significant higher than that in the control (P = 0.000). Compared with △FCa(2+) on the condition without treating with inhibitor of Na(+)-Ca(2+) exchanger, △FCa(2+) was 363 ± 21 in ADS group and △FCa(2+) was 237 ± 20 in control group after treatment. When compared with before treatment, there was no significant difference in both groups (P > 0.05).
The increase of intracellular Ca(2+) induced by estrogen at EMI smooth muscle cells in adenomyosis patients was mostly from the release of arcoplasmic reticulum, and also from the Ca(2+) influx controlled by L-type calcium channel. The increase of Ca(2+) inducing abnormal contraction of EMI muscle may have relationship with the development of adenomyosis.
探讨雌激素对子宫腺肌病患者子宫内膜 - 肌层界面（EMI）平滑肌细胞游离钙的调节机制。
2011年9月至2012年11月，收集59例行子宫切除术患者的子宫肌层标本，其中28例子宫腺肌病患者作为腺肌病（ADS）组，31例宫颈上皮内瘤变Ⅲ级患者作为对照组。培养EMI平滑肌细胞并加载钙离子荧光探针fluo - 4/AM。用三磷酸肌醇（IP3）受体拮抗剂、肌浆网钙 - 三磷酸腺苷（ATP）阻滞剂、兰尼碱受体操纵的Ca(2+)耗竭剂、L型钙通道抑制剂、钠 - 钙(2+)交换体抑制剂处理后，用雌激素刺激标记细胞。通过激光扫描显微镜检测细胞内Ca(2+)荧光强度的变化。细胞内Ca(2+)浓度的变化用△FCa(2+)表示。
（1）在正常钙条件下，雌激素刺激后，ADS组和对照组细胞内Ca(2+)荧光强度均高于未用雌激素处理组。ADS组△FCa(2+)为384±26，对照组△FCa(2+)为235±20。ADS组△FCa(2+)高于对照组（P<0.01）。在无钙条件下，ADS组△FCa(2+)为207±17，对照组△FCa(2+)为221±19。ADS组△FCa(2+)与对照组升高幅度相近（P = 0.731）。ADS组△FCa(2+)与有钙条件下相比显著降低（P<0.01）。而对照组有钙与无钙条件下差异无统计学意义（P = 0.060）。（2）用IP3受体拮抗剂、肌浆网钙 - ATP阻滞剂、兰尼碱受体操纵的Ca(2+)耗竭剂处理后，两组△F[Ca(2+)]i均显著降低（P<0.05），ADS组升高幅度显著高于对照组（P<0.05）。（3）用L型钙通道抑制剂处理后，ADS组△FCa(2+)为211±19，对照组△FCa(2+)为203±16，两组细胞内Ca(2+)均无显著升高（P>0.05）。但ADS组处理后△FCa(2+)较处理前显著降低（211±19 vs 384±28；P = 0.001）。对照组升高幅度与处理前相近（203±16 vs 234±22，P = 0.141）。（4）用钠 - 钙(2+)交换体抑制剂处理后，ADS组△FCa(2+)为357±24，对照组△FCa(2+)为209±19。ADS组升高幅度显著高于对照组（P = 0.000）。与未用钠 - 钙(2+)交换体抑制剂处理时相比，处理后ADS组△FCa(2+)为363±21，对照组△FCa(2+)为237±20。两组与处理前相比差异无统计学意义（P>0.05）。
子宫腺肌病患者EMI平滑肌细胞雌激素诱导的细胞内Ca(2+)升高主要源于肌浆网释放，也有L型钙通道控制的Ca(2+)内流。Ca(2+)升高诱导EMI肌层异常收缩可能与子宫腺肌病的发生发展有关。
