【作者】 薛婧；

【导师】 温泽清；

【作者基本信息】 山东大学 ， 妇产科学， 2013， 博士

【摘要】 子宫腺肌病是普通妇科领域的常见良性疾病,不会导致患者死亡,但是该病发病率高,所引起的一系列不适,严重影响了患者的生存质量。在妇科门诊中,子宫腺肌病是导致月经过多,痛经和性交痛的最常见原因之一,也是引起不孕不育的常见原因,目前除手术外仍未发现有效的治疗方法。腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)是目前研究较多的,在细胞内与能量代谢调节相关的蛋白激酶。由α,β,γ三种亚单位构成。其中α-亚单位为催化单位,β-亚单位和γ-亚单位为两个调节单位。当细胞受到应急刺激,导致ATP生成减少或消耗增加,AMP/ATP比值升高时,AMPK将被激活。活化后的AMPK,通过糖原合成的增加(例如糖摄取的增加,糖酵解和脂肪酸氧化等),并抑制消耗糖原的相关细胞活动(如脂肪酸,胆固醇和蛋白质的合成等),从而增加细胞内的ATP水平。磷脂酰肌醇-3-羟基激酶(phosphatidylinositol-3-ki-nase,PI3K)及丝氨酸／苏氨酸蛋白激酶Akt又称PKB(protein kinase B)在细胞存活和凋亡中起重要作用。PI3K是细胞生命活动中重要的调节分子,Akt是该通路的中心环节,其功能涉及血管生成、细胞周期调控、凋亡的启动、细胞侵袭性和端粒酶活性等诸多方面。以往诸多探究发现PI3K/Akt信号通路的激活与妇科疾病的发生、发展、浸润与转移密切相关,为妇科肿瘤的机制研究以及治疗方案提供了新的思路。通过对PI3K/Akt信号通路的不断深入研究,妇科肿瘤的发生发展机制也越来越清晰。但是目前就普通妇科良性疾病如子宫肌瘤、子宫腺肌病等的发病机制研究以及是否有PI3K/AKT通路参与有待进一步探讨。已有研究证实,AMPK信通路号与PI3K通路之间存在密切的联系。在PI3K异常活化相关的疾病中,例如癌症,AMPK已经成为一个新的潜在的治疗靶点。然而,迄今为止,还没有研究对AMPK在子宫腺肌病的发病机制中的作用进行研究探讨。二甲双胍是AMP-活化蛋白激酶激活剂,广泛用于治疗2型糖尿病。最近,二甲双胍已成为治疗女性多囊卵巢综合征(PCOS)的代表性药物。二甲双胍已经被证实能改善多囊卵巢综合征患者的生育异常,恢复排卵,提高生育能力。尽管二甲双胍的治疗能使多囊卵巢综合征患者获益,然而其对子宫内膜的作用还有待探索。鉴于AMPK和PI3K/AKT信号转导通路之间的关系,我们推测二甲双胍可能是一个新AKT通路抑制剂。因此,为了更好地了解二甲双胍在妇科领域的治疗潜力,我们研究的目标是在体外评估二甲双胍对子宫内膜间质细胞增殖的影响,并初步探讨该作用的可能机制。第一部分：AMPK蛋白在子宫内膜间质细胞中的表达研究目的1、检测AMPK在正常子宫内膜间质细胞(N-ESCs)及腺肌病在位子宫内膜间质细胞(A-ESCs)中是否表达及表达差异。2、检测AMPK特异性阻断剂Compound C对N-ESCs、A-ESCs中AMPK的表达的影响3、探讨Compound C用于阻断AMPK通路的条件研究方法1、收集无菌状态的正常子宫内膜及腺肌病在位子宫内膜标本,消化分离获得间质细胞2、应用免疫细胞化学方法(IHC)检测N-ESCs、A-ESCs中AMPK的表达3、应用蛋白质免疫印迹(Western Blot)方法,检测AMPK在N-ESCs、A-ESCs中的表达差异。4、将AMPK特异性阻断剂Compound C分别作用于N-ESCs和A-ESCs,采用Western Blot方法检测磷酸化AMPK(p-AMPK)表达差异结果1、AMPK在所检测正常子宫内膜及腺肌病在位子宫内膜间质细胞中均有表达。计算两组细胞评分,A-ESCs组30张切片评分值为3.4±0.1,N-ESCs组17张切片评分为2.2±0.2。腺肌病组约为正常组1.5倍(p<0.05)。2、Western Blot分析结果表明A-ESCs中AMPK蛋白的相对表达量(AMPK/β-actin)为83.4%±13.8%,高于N-ESCs中相对表达量58.9%±9.8%。腺肌病组的AMPK相对表达量是正常组的1.4倍(p<0.001)。3、应用Compound C作用1h后检测各组细胞中p-AMPK/AMPK,在A-ESCs、N-ESCs中的平均抑制效应分别为2.4±0.5倍和2.9±0.9倍。在A-ESCs、N-ESCs中应用Compound C1h和24h的平均抑制效应均无差别(p>0.5)。结论AMPK在正常子宫内膜及腺肌病在位子宫内膜间质细胞中均有表达,且在腺肌病在位子宫内膜间质细胞(A-ESCs)中表达强于正常子宫内膜间质细胞(N-ESCs)中表达;将AMPK特异性阻断剂Compound C分别作用于N-ESCs和A-ESCs后,检测到的磷酸化AMPK(p-AMPK)表达降低,表明Compound C阻断AMPK通路。第二部分：二甲双胍调节子宫腺肌病在位子宫内膜基质细胞生长的作用及其机制研究研究目的1、检测二甲双胍对正常子宫内膜间质细胞(N-ESCs)及腺肌病在位子宫内膜间质细胞(A-ESCs)生长增殖的作用2、探讨二甲双胍对A-ESCs生长增殖作用随月经周期变化的规律3、体外实验探讨二甲双胍对A-ESCs生长增殖作用的可能机制研究方法1、收集无菌状态的正常子宫内膜及腺肌病在位子宫内膜间质细胞,MTT法检测二甲双胍对其生长增殖的作用2、应用蛋白质免疫印迹(Western Blot)方法检测二甲双胍作用于A-ESCs对AMPK表达的影响3、应用蛋白质免疫印迹(Western Blot)方法和MTT法,检测二甲双胍对处于不同月经期的A-ESCs的作用,并进一步探讨该作用之间是否存在差异。4、应用蛋白质免疫印迹(Western Blot)方法探讨AMPK与Akt通路的关联关系结果1.二甲双胍可以抑制子宫内膜间质细胞的生长,抑制作用呈浓度依赖性.N-ESCs: IC507.87mmol/L;A-ESCs:IC502.45mmol/L。2.二甲双胍作用后A-ESCs中活化AMPK蛋白的相对表达量增加了2.0±0.3倍,Compound C可以阻断二甲双胍的作用。各实验组的磷酸化AMPK蛋白相对表达量即AMPK活化率分别为：空白对照组43.9%±1.6%,二甲双胍组83.2%±6.1%,Compound C组23.2%±5.2%,Compound C+二甲双胍组25.1%±4.1%。3.应用雌激素干预后增生期A-ESCspAMPK/AMPK减少2.1+0.8倍,分泌期减少2.5+0.5倍。4.二甲双胍作用下,增生期A-ESCs中活化AMPK蛋白的相对表达量为60.2±2.5(1.6倍于对照组),分泌期A-ESCs中活化AMPK蛋白的相对表达量为86.8±4.8(2.3倍于对照组)5.二甲双胍作用于增生期A-ESCs以及分泌期A-ESCs后检测磷酸化(活化)AKT蛋白的相对表达量分别减少2.3倍和3.2倍,该抑制作用与AMPK通路相关,Compound C阻断AMPK通路后,该抑制作用消失。结论二甲双胍对子宫内膜间质细胞的生长呈浓度依赖性抑制作用。二甲双胍通过活化AMPK通路抑制Akt通路达到抑制子宫腺肌病在位子宫内膜增长的作用,该抑制作用在增生期和分泌期子宫内膜间质细胞中均有表现,但在分泌期子宫内膜间质细胞中抑制作用表现更明显,并且在使用Compound C阻断AMPK通路后,该抑制作用消失。更多还原

【Abstract】 Part IThe expression of protein AMPK in endometrial stromal cellsObjective1. To investigate the expression of AMPK in normal endometrial stromal cells (N-ESCs) and adenomyotic eutopic endometrial stromal cells (A-ESCs)2. To detect the effect of Compound C to the expression level of AMPK in N-ESCs and A-ESCs.3. To explore the conditions of Compound C for blocking AMPK pathway.Methods1. Paired fresh normal endometria and adenomyotic eutopic endometria were collected from untreated patients subjected to radical hysterectomy. Then endometrial stromal cells were got by primary cell culture.2. Immunocytochemistry (IHC) was used to detect the purity of the ESCs and the expression of AMPK in N-ESCs and A-ESCs.3. Western blot (WB) assay was used to detect the AMPK protein level in N-ESCs and A-ESCs.4. With or without Compound C, we use Western blot assay to detect the expession of AMPK protein level in N-ESCs and A-ESCs.Result1. The purity of the ESCs was98%after the first and second passages. 2. AMPK was positive expressed in the cytoplasm of both N-ESCs and A-ESCs; A-ESCs exhibited greater AMPK expression than N-ESCs (P<0.05); AMPK expression levels (AMPK/β-actin) in A-ESCs and N-ESCs14groups were83.4%±13.8%and58.9%±9.8%, respectively (i.e.1.4-fold more).3. Compound C was found to significantly inhibit the phosphorylation of AMPK in a time-dependent manner. In both N-ESCs and A-ESCs, after1h exposure of Compound C, the average inhibition was2.4±0.5-fold2in A-ESCs and2.9±0.9-fold in N-ESCs.ConclusionAMPK was positive expressed in the cytoplasm of both N-ESCs and A-ESCs; A-ESCs showed stronger expression level than N-ESCs. Compound C can block AMPK pathway. Part II:The effect and the possible mechanism of metformin on the growth of A-ESCsObjective1. To evaluate the effect of metformin on the growth of endometrial stromal cells (ESCs).2. To compare the effect of metformin on A-ESCs variation with the menstrual cycle 3. To explore the possible mechanisms of metformin on A-ESCsMethods1. The functional effect of metformin on cell proliferation was evaluated using MTT assay.2. The application of Western blotting (Western Blot) was used to detect the effect of metformin on AMPK activation.3. Western Blot and MTT assay were used to explore the different effect during menstrual cycle.4. Western Blot was used to detect the relationship between AMPK and Akt pathway.Results1. Metformin can inhibit endometrial stromal cell growth inhibition in a dose dependent manner.N-ESCs:IC507.87mmol/L; A-ESCs:IC502.45mmol/L.2. Metformin was associated with a significant2.0±0.3-fold increase in AMPK activation (p-AMPK/AMPK) compared with controls (*P<0.001); The relative activated AMPK levels in A-ESCs in control, metformin, compound C, and compound C+metformin treated groups were43.9%±1.6%,83.2%±.1%,23.2%±5.2%, and25.1%±4.1%,3. Metformin inhibited the growth of A-ESCs from proliferative phase by47.8%±1.2%and cells from secretory phase by58.0%±0.9%relative to controls. The effects of metformin on activating AMPK18signaling (p-AMPK/AMPK) were obviously(*P<0.001).The relative activated AMPK levels in cells during the secretory phase and proliferative phase1were86.8±4.8(2.3-fold increase versus control),60.2±2.5(1.6-fold increase versus2control), respectively.4. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were pronounced in A-ESCs from secretory phase (3.2-fold14inhibition versus control) than that from proliferation phase (2.3fold inhibition versus control).ConclusionMetformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting more effective of metformin on A-ESCs from secretory phase.更多还原