米非司酮对人早孕绒毛中妊娠相关因子的影响
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摘要
目的:米非司酮是一种合成类固醇,其结构类似炔诺酮,是孕激素受体的拮抗剂。较早的研究认为,米非司酮使蜕膜发生变性、坏死,胚胎死亡而终止妊娠。随着研究的深入发现米非司酮可直接作用于绒毛滋养层细胞。近年研究表明,着床期的胚胎和子宫内膜除直接受到雌、孕激素的调节外,胚胎和子宫内膜自身还合成和分泌多种着床相关的蛋白因子,如血管内皮生长因子、白血病抑制因子、表皮生长因子、整合素、骨桥蛋白及基质金属蛋白酶等,它们以自分泌或旁分泌方式协调母胎的特殊生理状态。胚泡着床及妊娠成功的建立至少需要经过以下三个过程:(1)胚泡和细胞外基质的粘附;(2)着床部位细胞外基质的降解;(3)穿透细胞外基质。其中第一和第三个过程需要基质蛋白的细胞表面受体如整合素和骨桥蛋白等的参与,第二过程则需要能够降解细胞外基质的相应酶类,如基质金属蛋白酶等的参与。任何影响胚胎植入过程的因素,将导致植入的失败。米非司酮作为孕激素受体拮抗剂,不仅影响孕酮的生物学效应及多种蛋白因子,对妊娠及其产物的代谢和形态结构的影响也是多方面的,但是对于其调节机制尚不十分清楚。另外,目前国内外报道的米非司酮对早孕绒毛中骨桥蛋白及基质金属蛋白酶表达的研究结果也不完全一致。
本研究采用免疫组织化学法和荧光定量PCR,研究米非司酮对早孕绒毛中孕激素受体、雌激素受体、骨桥蛋白、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)表达的影响,进一步探讨米非司酮抗早孕机制。
材料和方法:将20名要求人工流产的正常妇女随机分为两组,其中10名妇女给予米非司酮150mg,服药后12～24h行负压吸宫术(研究组),另外10名妇女直接行负压吸宫术(对照组)。负压吸宫术后,即刻收集绒毛组织。采用荧光实时定量聚合酶链反应方法检测两组绒毛组织中雌激素受体和孕激素受体的基因表达;采用免疫组织化学法检测两组早孕绒毛组织的骨桥蛋白、MMP-2和MMP-9的蛋白表达。
结果:HE染色结果显示,对照组可见正常早孕绒毛组织形态学;研究组为部分绒毛间质水肿,绒毛组织呈小灶性变性、坏死,炎症细胞浸润;少数细胞核固缩、破碎、细胞质不均匀及空泡变性。免疫组织化学结果显示,骨桥蛋白、MMP-2和MMP-9的阳性表达产物为棕黄色颗粒,主要位于细胞膜和(或)细胞质内,也可见于细胞核上。对照组早孕绒毛组织中合体滋养层细胞、细胞滋养层细胞都显现骨桥蛋白、MMP-2和MMP-9的表达。在绒毛组织中相应部位,研究组骨桥蛋白、MMP-2和MMP-9的表达强度明显减弱。荧光实时定量聚合酶链反应结果表明,研究组绒毛孕激素受体mRNA的表达显著高于对照组(P＜0.01);两组绒毛雌激素受体mRNA的表达差异无统计学意义(P＞0.05)。
结论:1.正常早孕绒毛组织结构完整,米非司酮作用后结构发生改变,绒毛组织变性坏死。表明米非司酮可直接抑制绒毛滋养细胞增生,促进其变性、坏死,从而达到抗早孕的效果。2.服用米非司酮后,早孕绒毛中孕激素受体mRNA的表达量显著上调,而雌激素受体mRNA的表达量无显著变化。这一结果提示米非司酮通过与孕激素受体结合,竞争性拮抗孕激素活性,干扰了雌激素受体、孕激素受体之间的动态平衡,这可能是绒毛组织变性坏死的主要原因。3.研究组早孕绒毛骨桥蛋白的阳性表达显著低于对照组。这一结果提示米非司酮抑制了早孕绒毛中骨桥蛋白的合成,可能使滋养层细胞和蜕膜细胞的黏附、增殖和迁移能力下降,影响了胚泡着床和胎盘的形成,导致流产。4.研究组早孕绒毛MMP-9和MMP-2的阳性表达显著低于对照组。这一结果提示米非司酮抑制了早孕绒毛MMP-9和MMP-2的合成,使绒毛功能减退,难以维持妊娠,最终流产。
Objective: The mifepristone is a synthesized steroid, its structure is similar to the norethisterone, and it has the characteristics of the progesterone receptor antagonist. Early research showed that mifepristone could induce apoptosis and necrosis in deciduas, and lead to termination of pregnancy. The further investigation found that the mifepristone could directly affect the chorionic nutrient cells. The recent studies showed that the fine regulation of estrogen and progesterone was necessary to embryo and endometrium, both embryo and endometrium themselves also secreted many kinds of protein factors, such as vascular endothelial growth factor (VEGF), leukaemia inhibitory factor (LIF), epidermal growth factor (EGF), integrins, osteopontin (OPN), matrix metalloproteinases (MMPs) and so on. These protein factors acted as autocrine or paracrine manner to co-regulate the specific physiological state of pregnancy. The fine regulation between embryo and maternal environment was required for the success of embryo implantation and pregnancy establishment, which involved a series of cellular or molecular events, requiring various modulating factors, among which including integrins, osteopontin, MMPs and so forth. Disturbance of the factors affected the process of embryo implantation could lead to the failure of implantation. Mifepristone, an antagon of the progesterone receptor (PR), is able to influence various protein factors and the biological effect of the progesterone. Mifepristone has great impact on the gestation as well as the metabolism and morphosis of its products. The regulatory mechanism of mifepristone is still not clear. Besides, the research results about the expression of OPN and MMPs in the chorion from early pregnancy under the mifepristone influence are inconsistency.
In the present study, we used the immunohistochemistry and Real-time quantitative PCR to detect the expression of PR, estrogen receptor (ER), OPN, MMP-2 and MMP-9 after administration of the mifepristone during the early pregnant stage in order to further explore the mechanism of mifepristone-induced abortion.
Materials and methods: Twenty normal women requesting abortion were randomized to study and control groups. Ten Women in the study group took 150mg of mifepristone 12～24h before vacuum aspiration. Another ten women in the control group experienced directly vacuum aspiration. The foetal sac was immediately collected from vacuum aspiration of the uterus without prior dilatation of the cervix. Real-time quantitative PCR was used to detect the gene expression of estrogen receptor and progesterone receptor in the chorion from the women of both groups. Meanwhile the protein expressions of OPN, MMP-2 and MMP-9 in the chorion were detected with immunohistochemistry method.
Results: The results of HE staining showed that the histomorphology of the early pregnant chorial cells were normal in the control group. In the study group the interstitial edema was found in some chorion. Furthermore the patchy degeneration, the necrosis and the inflammatory cells infiltration were found in the chorion. The karyopyknosis, the quassation, the uneven cytoplasm, the vacuolar degeneration were also seen in a few cells. The results of the immunohistochemistry showed that the OPN, MMP-2 and MMP-9 were located in the cytoplasm and/or on the cell membrane and/or in the nucleus. In control group, the OPN, MMP-2 and MMP-9 were expressed in the syncytiotrophoblast cell and cytotrophoblast cell of the chorion during the early pregnant stage. In the study group, the OPN, MMP-2 and MMP-9 were weakly expressed in the homologus position. The result of Real-time quantitative PCR indicated that the expression of PR mRNA in the chorion in the study group was significantly higher than that in the control group(P< 0.01). There was no statistical differences of the expression of ER mRNA in the chorion between the two groups(P>0.05).
Conclusion: 1. The normal chorion tissue of early pregnancy had complete structure. It was changed after administration of mifepristone. The chorion cell turned to dropsy, degenerate and necrosis. It indicates that mifepristone can directly repress the chorion nutrient cell, promote the necrosis and amotic, which may be the mechanism of termination of early pregnancy by mifepristone. 2. The expression of PR mRNA in the chorion in the study group was significantly higher than that in the control group, but there was no statistical difference of the expression of ER mRNA in the chorion between the two groups. The results suggest that the mifepristone competes the progestagen action by the combination with PR, inducing the decidua and chorion tissue degenerate and necrosis. The dynamic balance between ER and PR was disturbed and the pregnancy is hard to maintain, then inducing abortion. 3. The results of the immunohistochemistry showed that the expression of the OPN in the study group was much less than that in the control group. The results suggest that the mifepristone inhibits the synthesis of the OPN during the early pregnant stage; it may result in depressing the capability of the adhesion, the proliferation and the migration of the trophocyte and the decidual cells. So both the imbedding of the blastocyst and the placental formation are affected, then the abortion occurs. 4. The results of the immunohistochemistry showed that the expressions of the MMP-9 and MMP-2 in the study group were much less than that in the cnntrol group. The results suggest that the mifepristone inhibits the synthesis of the MMP-9 and MMP-2 during the early pregnant stage. So the normal function of the chorion decreases, the pregnancy is ended.
本研究采用免疫组织化学法和荧光定量PCR,研究米非司酮对早孕绒毛中孕激素受体、雌激素受体、骨桥蛋白、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)表达的影响,进一步探讨米非司酮抗早孕机制。
材料和方法:将20名要求人工流产的正常妇女随机分为两组,其中10名妇女给予米非司酮150mg,服药后12～24h行负压吸宫术(研究组),另外10名妇女直接行负压吸宫术(对照组)。负压吸宫术后,即刻收集绒毛组织。采用荧光实时定量聚合酶链反应方法检测两组绒毛组织中雌激素受体和孕激素受体的基因表达;采用免疫组织化学法检测两组早孕绒毛组织的骨桥蛋白、MMP-2和MMP-9的蛋白表达。
结果:HE染色结果显示,对照组可见正常早孕绒毛组织形态学;研究组为部分绒毛间质水肿,绒毛组织呈小灶性变性、坏死,炎症细胞浸润;少数细胞核固缩、破碎、细胞质不均匀及空泡变性。免疫组织化学结果显示,骨桥蛋白、MMP-2和MMP-9的阳性表达产物为棕黄色颗粒,主要位于细胞膜和(或)细胞质内,也可见于细胞核上。对照组早孕绒毛组织中合体滋养层细胞、细胞滋养层细胞都显现骨桥蛋白、MMP-2和MMP-9的表达。在绒毛组织中相应部位,研究组骨桥蛋白、MMP-2和MMP-9的表达强度明显减弱。荧光实时定量聚合酶链反应结果表明,研究组绒毛孕激素受体mRNA的表达显著高于对照组(P＜0.01);两组绒毛雌激素受体mRNA的表达差异无统计学意义(P＞0.05)。
结论:1.正常早孕绒毛组织结构完整,米非司酮作用后结构发生改变,绒毛组织变性坏死。表明米非司酮可直接抑制绒毛滋养细胞增生,促进其变性、坏死,从而达到抗早孕的效果。2.服用米非司酮后,早孕绒毛中孕激素受体mRNA的表达量显著上调,而雌激素受体mRNA的表达量无显著变化。这一结果提示米非司酮通过与孕激素受体结合,竞争性拮抗孕激素活性,干扰了雌激素受体、孕激素受体之间的动态平衡,这可能是绒毛组织变性坏死的主要原因。3.研究组早孕绒毛骨桥蛋白的阳性表达显著低于对照组。这一结果提示米非司酮抑制了早孕绒毛中骨桥蛋白的合成,可能使滋养层细胞和蜕膜细胞的黏附、增殖和迁移能力下降,影响了胚泡着床和胎盘的形成,导致流产。4.研究组早孕绒毛MMP-9和MMP-2的阳性表达显著低于对照组。这一结果提示米非司酮抑制了早孕绒毛MMP-9和MMP-2的合成,使绒毛功能减退,难以维持妊娠,最终流产。
Objective: The mifepristone is a synthesized steroid, its structure is similar to the norethisterone, and it has the characteristics of the progesterone receptor antagonist. Early research showed that mifepristone could induce apoptosis and necrosis in deciduas, and lead to termination of pregnancy. The further investigation found that the mifepristone could directly affect the chorionic nutrient cells. The recent studies showed that the fine regulation of estrogen and progesterone was necessary to embryo and endometrium, both embryo and endometrium themselves also secreted many kinds of protein factors, such as vascular endothelial growth factor (VEGF), leukaemia inhibitory factor (LIF), epidermal growth factor (EGF), integrins, osteopontin (OPN), matrix metalloproteinases (MMPs) and so on. These protein factors acted as autocrine or paracrine manner to co-regulate the specific physiological state of pregnancy. The fine regulation between embryo and maternal environment was required for the success of embryo implantation and pregnancy establishment, which involved a series of cellular or molecular events, requiring various modulating factors, among which including integrins, osteopontin, MMPs and so forth. Disturbance of the factors affected the process of embryo implantation could lead to the failure of implantation. Mifepristone, an antagon of the progesterone receptor (PR), is able to influence various protein factors and the biological effect of the progesterone. Mifepristone has great impact on the gestation as well as the metabolism and morphosis of its products. The regulatory mechanism of mifepristone is still not clear. Besides, the research results about the expression of OPN and MMPs in the chorion from early pregnancy under the mifepristone influence are inconsistency.
In the present study, we used the immunohistochemistry and Real-time quantitative PCR to detect the expression of PR, estrogen receptor (ER), OPN, MMP-2 and MMP-9 after administration of the mifepristone during the early pregnant stage in order to further explore the mechanism of mifepristone-induced abortion.
Materials and methods: Twenty normal women requesting abortion were randomized to study and control groups. Ten Women in the study group took 150mg of mifepristone 12～24h before vacuum aspiration. Another ten women in the control group experienced directly vacuum aspiration. The foetal sac was immediately collected from vacuum aspiration of the uterus without prior dilatation of the cervix. Real-time quantitative PCR was used to detect the gene expression of estrogen receptor and progesterone receptor in the chorion from the women of both groups. Meanwhile the protein expressions of OPN, MMP-2 and MMP-9 in the chorion were detected with immunohistochemistry method.
Results: The results of HE staining showed that the histomorphology of the early pregnant chorial cells were normal in the control group. In the study group the interstitial edema was found in some chorion. Furthermore the patchy degeneration, the necrosis and the inflammatory cells infiltration were found in the chorion. The karyopyknosis, the quassation, the uneven cytoplasm, the vacuolar degeneration were also seen in a few cells. The results of the immunohistochemistry showed that the OPN, MMP-2 and MMP-9 were located in the cytoplasm and/or on the cell membrane and/or in the nucleus. In control group, the OPN, MMP-2 and MMP-9 were expressed in the syncytiotrophoblast cell and cytotrophoblast cell of the chorion during the early pregnant stage. In the study group, the OPN, MMP-2 and MMP-9 were weakly expressed in the homologus position. The result of Real-time quantitative PCR indicated that the expression of PR mRNA in the chorion in the study group was significantly higher than that in the control group(P< 0.01). There was no statistical differences of the expression of ER mRNA in the chorion between the two groups(P>0.05).
Conclusion: 1. The normal chorion tissue of early pregnancy had complete structure. It was changed after administration of mifepristone. The chorion cell turned to dropsy, degenerate and necrosis. It indicates that mifepristone can directly repress the chorion nutrient cell, promote the necrosis and amotic, which may be the mechanism of termination of early pregnancy by mifepristone. 2. The expression of PR mRNA in the chorion in the study group was significantly higher than that in the control group, but there was no statistical difference of the expression of ER mRNA in the chorion between the two groups. The results suggest that the mifepristone competes the progestagen action by the combination with PR, inducing the decidua and chorion tissue degenerate and necrosis. The dynamic balance between ER and PR was disturbed and the pregnancy is hard to maintain, then inducing abortion. 3. The results of the immunohistochemistry showed that the expression of the OPN in the study group was much less than that in the control group. The results suggest that the mifepristone inhibits the synthesis of the OPN during the early pregnant stage; it may result in depressing the capability of the adhesion, the proliferation and the migration of the trophocyte and the decidual cells. So both the imbedding of the blastocyst and the placental formation are affected, then the abortion occurs. 4. The results of the immunohistochemistry showed that the expressions of the MMP-9 and MMP-2 in the study group were much less than that in the cnntrol group. The results suggest that the mifepristone inhibits the synthesis of the MMP-9 and MMP-2 during the early pregnant stage. So the normal function of the chorion decreases, the pregnancy is ended.
引文
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