杨 眉,蒋春樊,哈春芳
·论著·
·专题研究·
米非司酮对子宫内膜异位症子宫内膜腺上皮细胞骨桥蛋白和基质金属蛋白酶9表达的影响
杨 眉,蒋春樊,哈春芳
背景目前子宫内膜异位症的治疗方法仍比较有限,而米非司酮在临床上可用于治疗子宫内膜异位症,但其具体机制尚未明确。目的探讨米非司酮对子宫内膜异位症子宫内膜腺上皮细胞中骨桥蛋白(OPN)、基质金属蛋白酶9(MMP-9)表达的影响。方法选取2014年8月—2015年3月宁夏医科大学总医院妇科因卵巢巧克力囊肿行卵巢囊肿剥除术或患侧附件切除术的子宫内膜异位症患者21例为研究对象。按照术前是否接受米非司酮治疗将其分为米非司酮组(11例)和对照组(10例)。获取所有患者在位、异位内膜组织,采用免疫组化法检测其中OPN、MMP-9表达水平;取对照组患者在位子宫内膜组织,培养子宫内膜腺上皮细胞,以不同剂量(0、0.1、1.0、10.0 mmol/L)米非司酮进行干预,采用反转录-聚合酶链式反应(RT-PCR)法检测子宫内膜腺上皮细胞中OPN、MMP-9表达水平。结果米非司酮组在位、异位内膜组织中OPN、MMP-9表达水平均低于对照组(P<0.05)。0.1 mmol/L、1.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平低于0 mmol/L(P<0.05);1.0 mmol/L、10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于0.1 mmol/L(P<0.05);10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于1.0 mmol/L(P<0.05)。结论米非司酮可明显下调子宫内膜异位症子宫内膜中OPN、MMP-9表达水平,从而抑制子宫内膜的侵袭、黏附以及种植性转移。
子宫内膜异位症;米非司酮;骨桥蛋白质;基质金属蛋白酶9
杨眉,蒋春樊,哈春芳.米非司酮对子宫内膜异位症子宫内膜腺上皮细胞骨桥蛋白和基质金属蛋白酶9表达的影响[J].中国全科医学,2016,19(24):2925-2929.[www.chinagp.net]
YANG M,JIANG C F,HA C F.Influence of mifepristone on the expression of osteopontin and matrix metalloproteinase 9 in the endometrium epithelial cells of patients with endometriosis[J].Chinese General Practice,2016,19(24):2925-2929.
子宫内膜异位症是一种妇科常见疾病,以子宫内膜的异位黏附、侵袭为特点[1-2]。本课题组前期研究证明,骨桥蛋白(OPN)及基质金属蛋白酶9(MMP-9)在子宫内膜异位症的发病过程中起关键性作用[3-4]。临床上米非司酮可以用于治疗子宫内膜异位症,但其具体机制尚未明确[5-6]。为此,本研究分析米非司酮对子宫内膜异位症子宫内膜腺上皮细胞中OPN、MMP-9表达的影响,以期进一步明确米非司酮治疗子宫内膜异位症的分子机制。
1.1纳入与排除标准纳入标准:(1)为Ⅳ期子宫内膜异位症;(2)处于月经分泌期。排除标准:合并重大内科疾病及有激素治疗史患者。
1.2研究对象选取2014年8月—2015年3月宁夏医科大学总医院妇科因卵巢巧克力囊肿行巧克力囊肿剥除术或患侧附件切除术的子宫内膜异位症患者21例为研究对象,年龄21~49岁,平均年龄(34.2±5.4)岁;术前给予米非司酮治疗(10 mg/d,1次/d,疗程3个月)11例(米非司酮组),未进行药物治疗10例(对照组)。子宫内膜异位症诊断经2位病理科医师复诊明确。本研究经宁夏医科大学总医院伦理委员会审批通过,患者均签署知情同意书。
1.3研究方法
1.3.1获取内膜组织术中行诊刮术获取在位、异位内膜组织。
1.3.2免疫组化法检测在位、异位内膜组织中OPN、MMP-9表达水平收集在位、异位内膜组织,用4%中性甲醛溶液固定,石蜡包埋后切片,二甲苯脱蜡,严格按照试剂说明书操作。加入小鼠抗人OPN及MMP-9单克隆抗体(美国Abcam公司,均为1∶100稀释),4 ℃孵育过夜。DAB显色,显微镜下等同条件下观察并拍照。应用Image-Pro Rlus 6.0图像分析系统对图像进
本研究价值:
目前子宫内膜异位症的治疗方法仍比较有限,米非司酮在临床上可用来治疗子宫内膜异位症,但其具体机制尚未明确。本研究证明,米非司酮可下调子宫内膜异位症子宫内膜中OPN、MMP-9表达水平,从而抑制子宫内膜的侵袭、黏附以及种植性转移。这有助于推动药物治疗子宫内膜异位症的新进展,为后续对子宫内膜异位症从机制上进行有效的药物干预、指导临床治疗和随访提供依据。
行分析,每张切片选取5个高倍镜(×400)视野,分别计算每高倍镜视野下的OPN、MMP-9的IOD值,即OPN、MMP-9表达水平。
1.3.3子宫内膜腺上皮细胞培养及药物干预取对照组患者在位子宫内膜组织,低温条件下迅速送往实验室,剪成1 cm3小块,胶原酶消化,采用差速离心法去除间质细胞,将纯度95%以上的子宫内膜腺上皮细胞进行培养。子宫内膜腺上皮细胞达到60%融合时,去除培养液,将细胞置于无血清的培养液中12 h。将培养液换为含不同剂量(0、0.1、1.0、10.0 mmol/L)米非司酮(美国Sigma公司)的DMEM/Ham′s F12培养液48 h,并加入活性炭,进一步去除胎牛血清。
1.3.4反转录-聚合酶链式反应(RT-PCR)法检测子宫内膜腺上皮细胞中OPN、MMP-9表达水平Trizol裂解细胞,检测并计算出RNA的浓度和纯度,反转录后进行PCR,条件为94 ℃预变性5 min,94 ℃变性30 s,56 ℃退火30 s,72 ℃延伸30 s,共40个循环,72 ℃再延伸7 min,4 ℃保存。OPN上游引物:5′-ACAGCCGTGGGAAGGACAGTTA-3′,下游引物:5′-CCTGACTATCAATCACATCGGAATG-3′;MMP-9上游引物:5′-AGTCCACCCTTGTGCTCTTCCC-3′,下游引物:5′-TCTGCCACCCGAGTGTAACCAT-3′;β-actin上游引物:5′-AGCGAGCATCCCCCAAAGTT-3′,下游引物:5′-GGGCACGAAGGCTCATCATT-3′。采用2-ΔΔCt法计算出OPN、MMP-9 mRNA表达水平[3]。
2.1米非司酮组与对照组在位、异位内膜组织中OPN、MMP-9表达水平比较米非司酮组在位、异位内膜组织中OPN、MMP-9表达水平均低于对照组,差异有统计学意义(P<0.05,见表1、图1~4)。
Table 1Comparison of expression levels of OPN and MMP-9 in the eutopic and ectopic endometrium of patients in mifepristone group and control group
组别例数在位内膜组织OPN MMP-9异位内膜组织OPN MMP-9对照组103.31±1.314.81±1.535.13±1.475.50±2.15米非司酮组111.25±0.712.89±1.382.09±1.283.27±2.04t值3.012.653.103.40P值0.0180.0210.0120.005
注:OPN=骨桥蛋白,MMP-9=基质金属蛋白酶9
注:A1~A3为对照组,B1~B3为米非司酮组
图1米非司酮组与对照组在位内膜组织中OPN的表达
Figure 1Expression of OPN in the eutopic endometrium of patients in mifepristone group and control group
注:A1~A3为对照组,B1~B3为米非司酮组
图2米非司酮组与对照组在位内膜组织中MMP-9的表达
Figure 2Expression of MMP-9 in the eutopic endometrium of patients in mifepristone group and control group
注:A1~A3为对照组,B1~B3为米非司酮组
图3米非司酮组与对照组异位内膜组织中OPN的表达
Figure 3Expression of OPN in the ectopic endometrium of patients in mifepristone group and control group
注:A1~A3为对照组,B1~B3为米非司酮组
图4米非司酮组与对照组异位内膜组织中MMP-9的表达
Figure 4Expression of MMP-9 in the ectopic endometrium of patients in mifepristone group and control group
2.2不同剂量米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平比较不同剂量米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平比较,差异有统计学意义(P<0.05)。0.1 mmol/L、1.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平低于0 mmol/L,差异有统计学意义(P<0.05);1.0 mmol/L、10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于0.1 mmol/L,差异有统计学意义(P<0.05);10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于1.0 mmol/L,差异有统计学意义(P<0.05,见表2)。
Table 2Comparison of mRNA expression levels of OPN and MMP-9 in endometrium epithelial cells that intervened with mifepristone at the different doses
米非司酮(mmol/L)OPNmRNAMMP-9mRNA01.001.000.10.56±0.13a0.48±0.16a1.00.70±0.19ab0.75±0.21ab10.00.96±0.25bc1.05±0.26bcF值66.3042.75P值<0.001<0.001
注:与0 mmol/L比较,aP<0.05;与0.1 mmol/L比较,bP<0.05;与1.0 mmol/L比较,cP<0.05
目前关于子宫内膜异位症的研究多集中在内膜异位黏附、种植的具体机制,但仍不十分清楚[7]。众多研究显示,OPN在子宫内膜异位症的发病过程中可诱导细胞的侵袭、黏附、抗凋亡能力增强,并促进血管生成[3]。而MMP-9是分子量最大的一种明胶酶,能降解细胞外基质,促进血管内皮细胞出芽并表达于细胞膜表面,促使细胞向周围浸润、侵袭和转移[3,8]。目前对子宫内膜异位症的药物治疗主要是甾体激素类药物,如米非司酮,但其具体机制尚未明确[5-6,9-10]。本研究组前期论证了雌激素调节OPN/MMP-9通路导致子宫内膜异位症腺上皮细胞侵袭性增强,可能是子宫内膜异位症发病的重要因素[3]。米非司酮是一种抗孕酮和抗糖皮质激素的甾体类药物,那么米非司酮对子宫内膜异位症腺上皮细胞中OPN、MMP-9的表达起到什么样的作用呢?
本研究结果显示,米非司酮组在位、异位内膜组织中OPN、MMP-9表达水平均低于对照组,0.1 mmol/L、1.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平低于0 mmol/L,1.0 mmol/L、10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于0.1 mmol/L,10.0 mmol/L米非司酮干预子宫内膜腺上皮细胞中OPN、MMP-9 mRNA表达水平高于1.0 mmol/L,提示米非司酮可抑制子宫内膜异位症腺上皮细胞OPN、MMP-9表达,其最佳干预剂量为0.1 mmol/L。米非司酮为19-去甲类固醇、炔诺酮的衍生物,通过与其受体竞争性结合而发挥作用,在临床上已广泛用于抗早孕、中孕引产、软化宫颈等,亦可用于子宫内膜异位症、子宫腺疾病的治疗[11-13]。本研究组前期的动物实验发现,米非司酮可降低子宫内膜异位症模型大鼠在位、异位内膜中OPN、MMP-9表达水平[14]。而其他学者的研究显示,米非司酮可竞争性结合下丘脑、垂体组织中的孕激素受体,使卵巢功能受抑制,出现闭经现象,进一步导致异位的子宫内膜萎缩[15]。米非司酮通过直接抑制雌、孕激素受体的合成从而阻断内膜对雌、孕激素的反应性,使异位内膜萎缩[16]。米非司酮可通过抑制异位内膜细胞白介素6、纤维母细胞生长因子、血管内皮生长因子的合成,提升异位内膜中人第10号染色体缺失的磷酸酶(PTEN)、B淋巴细胞瘤-2基因、Fas表达水平,抑制异位内膜的生长并促进其凋亡[17-18]。米非司酮可通过作用于下丘脑、垂体、子宫内膜等多水平抑制在位、异位内膜对雌激素的反应,从而下调在位、异位内膜中OPN、MMP-9表达水平。
本研究从组织及细胞层面验证了米非司酮对子宫内膜异位症子宫内膜OPN、MMP-9表达的抑制作用,但其对子宫内膜异位症的治疗机制可能是多层面的,OPN、MMP-9表达的下调只是多种机制之一。另外,米非司酮可抑制雌、孕激素受体的合成,其是否造成子宫内膜异位症腺上皮细胞雌、孕激素受体比例变化,其与子宫内膜异位症腺上皮细胞受体结合的具体机制如何,均不明确,有待进一步研究。
综上所述,米非司酮可明显下调子宫内膜异位症子宫内膜中OPN、MMP-9表达水平,从而抑制子宫内膜的侵袭、黏附以及种植性转移,这可能也是米非司酮治疗子宫内膜异位症的重要机制。
作者贡献:杨眉进行试验设计与实施、资料收集整理、撰写论文、成文并对文章负责;杨眉、蒋春樊进行试验实施、评估、资料收集;哈春芳进行质量控制及审校。
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(本文编辑:崔丽红)
Influence of Mifepristone on the Expression of Osteopontin and Matrix Metalloproteinase 9 in the Endometrium Epithelial Cells of Patients With Endometriosis
YANGMei,JIANGChun-fan,HAChun-fang.
DepartmentofObstetricsandGynecology,XiangyangCentralHospital,AffiliatedHospitalofHubeiUniversityofArtsandScience,Xiangyang441021;NingxiaMedicalUniversity,Yinchuan750004,ChinaCorrespondingauthor:HAChun-fang,DepartmentofGynecology,GeneralHospitalofNingxiaMedicalUniversity;KeyLaboratoryofFertilityPreservationandMaintenanceoftheMinistryofEducation,Yinchuan750004,China;E-mail:hachunfang@163.com
BackgroundThe therapeutic method towards endometriosis is still in limitation,and mifepristone can be used to treat endometriosis in clinic,but its specific mechanism still remains unclear.ObjectiveTo investigate the influence of miferpristone on the expression of osteopontin (OPN) and matrix metalloproteinase 9 (MMP-9) in the endometrium epithelial cells of patients with endometriosis.Methods21 patients with endometriosis,who suffered ovarian cyst enucleation or adnexectomy due to ovarian chocolate cyst in Department of Gynecology in General Hospital of Ningxia Medical University from August 2014 to March 2015,were recruited in the study.They were divided into mifepristone group (11 cases) and control group (10 cases) based on whether they had undergone mifepristone therapy.The eutopic and ectopic endometrium of all these patients were obtained through surgery,and immunohistochemistry method was used to detect the expression level of OPN and MMP-9;the culture of endometrium epithelial cells in the eutopic endometrium of patients in control group were performed with the intervention of different doses of mifepristone (0,0.1,1.0,10.0 mmol/L),and inverse transcription-polymerase chain reaction (RT-PCR) method was used to investigate the expression level of OPN and MMP-9 in the endometrial epithelial cells.ResultsThe expression levels of OPN and MMP-9 in mifepristone group both in eutopic and ectopic endometrium were lower than those in control group (P<0.05).The mRNA expression levels of OPN and MMP-9 in endometrium epithelial cells after the intervention of mifepristone at a dose of 0.1 and 1.0 mmol/L were lower than those at 0 mmol/L (P<0.05);the mRNA expression levels of OPN and MMP-9 in endometrium epithelial cells after the intervention of mifepristone at a dose of 1.0 and 10.0 mmol/L were higher than those at 0.1 mmol/L (P<0.05);the mRNA expression levels of OPN and MMP-9 in endometrium epithelial cells after the intervention of mifepristone at the dose of 10.0 mmol/L were higher than those at 1.0 mmol/L (P<0.05).ConclusionMifepristone can obviously lower the expression levels of OPN and MMP-9 in endometrium of endometriosis,and thus inhibit the invasion,adhesion and implantation metastasis of endometrium.
Endometriosis;Mifepristone;Osteopontin;Matrix metalloproteinase 9
国家自然科学基金资助项目(81160078)
441021 湖北省襄阳市中心医院 湖北文理学院附属医院妇产科(杨眉),病理科(蒋春樊);宁夏医科大学(杨眉);宁夏医科大学总医院妇科,生育力保持重点实验室(哈春芳)
哈春芳,750004 宁夏银川市,宁夏医科大学总医院妇科,生育力保持重点实验室;E-mail:hachunfang@163.com
R 711.71
ADOI:10.3969/j.issn.1007-9572.2016.24.011
2016-03-05;
2016-07-05)