【作者】 任月芳；

【导师】 郑伟；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 子宫腺肌病是正常位置的子宫内膜腺体和间质向肌壁内良性侵入的一种妇科疾病,伴随周围肌层的代偿性肥大和增生。发病率不断上升,多发生于40-50岁的经产妇女,其典型症状为盆腔疼痛和异常子宫出血。目前多数研究者认为子宫腺肌病是子宫内膜基底层向肌层内陷生长、迁移播散的结果。保守治疗包括子宫动脉栓塞、激素治疗(孕激素、持续口服避孕药、抗雌激素制剂和GnRHa)和超声聚焦等,但效果有限。子宫切除术是严重子宫腺肌病患者的选择和最终明确诊断的方法。子宫腺肌病的病因、发病机制和更好的保守治疗手段目前仍不明了,需进一步探究。自噬以自噬体和自噬溶酶体的聚集为特征,被认为是一种区别于凋亡的Ⅱ型程序性细胞死亡。Beclin 1是酵母自噬相关基因的哺乳细胞同源基因,Beclin 1基因定位于染色体17q21,是自噬的直接执行者。在75%的卵巢癌,50%的乳腺癌和40%的前列腺癌中等位基因缺失,已被确定为一个新的候选抑癌基因。Beclin 1蛋白是自噬与凋亡重要的汇合点。Beclin 1具有BH3结构域,中央卷曲螺旋结构域(CCD)和进化上保守的结构域(ECD)。Bcl-2与Beclin 1的BH3结构域相互作用。缺氧诱导因子-1α(HIF-1α)是细胞应激缺氧时所产生的转录激活因子。研究显示HIF-1α在很多正常组织中不表达,而在人类肿瘤中高表达。HIF-1α激活代谢和致病途径,与肿瘤的血管生成、生长、侵袭和转移有关。HIF-1α在恶性肿瘤进展中的功能可分为以下几种：(1)促进肿瘤细胞离散；(2)促进自分泌移动因子及各种生长因子的表达；(3)提高基质金属蛋白酶家族的表达,促进肿瘤细胞对细胞外间质的降解而发生侵袭；(4)促进新生血管生成。可见缺氧贯穿于肿瘤整个生长过程,赋予肿瘤细胞不断生长、恶性演进和转移的潜能,HIF-1α与肿瘤的发生、发展密切相关。研究显示自噬与缺氧相关。缺氧能诱导拥有完整凋亡机制的肿瘤细胞发生自噬性细胞死亡。在小鼠胚胎成纤维细胞中,HIF-1诱导BNIP3表达,其中BNIP3能激发线粒体发生选择性的自噬,这个过程需要HIF-1依赖的BNIP3的表达和Beclin 1的固有表达,延长细胞缺氧的时间后,线粒体自噬是阻止活性氧过多产生和细胞死亡增加的适应性代谢反应。细胞缺氧培养24小时后BNIP3和BNIP3L表达上调,而流式细胞技术检测不到凋亡,由此显示BNIP3和BNIP3L能促进细胞自噬而不是凋亡。有研究显示Beclin 1和HIF-1a是重要的预后因子。目前尚未见Beclin 1与子宫腺肌病发生发展关系的报道及Beclin 1与HIF-1a在子宫腺肌病相关性的报道。为明确Beclin 1在子宫腺肌病发生发展中的作用及缺氧对其的影响,本文利用免疫组化、RT-PCR.蛋白免疫印迹和细胞培养等技术从以下几个方面进行研究：1. Beclin 1在子宫腺肌病在位内膜中的表达及其临床意义；2. Beclin 1和HIF-1α在子宫腺肌病在位内膜、病灶中的表达、临床意义及相关性研究；3. Beclin 1在子宫腺肌病在位内膜间质细胞中的表达及缺氧对其的影响。第一部分Beclin 1在子宫腺肌病在位内膜中的表达及其临床意义目的：探讨自噬相关基因Beclin 1在子宫腺肌病在位内膜中的表达及其与临床特征的相关性。方法：采用RT-PCR和蛋白免疫印迹技术检测30例子宫腺肌病在位内膜组织中Beclin 1 mRNA及蛋白的表达,并以32例正常子宫内膜组织作为对照。结果：1.通过灰度分析,子宫腺肌病组Beclin 1 mRNA相对表达量为(0.71±0.10),明显低于对照组(0.93±0.10)(P<0.001)。子宫腺肌病组Beclin 1蛋白相对表达量为(0.82±0.22),明显低于对照组(1.22±0.36)(P<0.001)。2.内膜Beclin 1蛋白表达水平与血清中CA125水平呈负相关(n=62,r2=0.094,P=0.015),与盆腔疼痛亦呈负相关(n=62,r2=0.293,P=0.000)。第二部分Beclin 1和HIF-1α在子宫腺肌病在位内膜、病灶中的表达、临床意义及相关性研究目的：探讨Beclin 1和HIF-1α在子宫腺肌病在位内膜、病灶中的表达、临床意义及其相关性。方法：采用免疫组化方法检测53例子宫腺肌病在位内膜组织中Beclin 1和HIF-1α的表达,并以57例正常子宫内膜组织作为对照,同时检测30例病灶中Beclin1和HIF-1α的表达。结果：1. Beclin 1蛋白在子宫内膜组织标本中腺细胞和间质细胞中均有表达,其表达主要见于细胞浆内。HIF-1α蛋白在子宫内膜组织标本中腺细胞和间质细胞中均有表达,其表达细胞浆、细胞核均有表达,在腺肌病病灶中以腺上皮表达为主,其表达主要见于细胞浆内。2.在位腺肌病组Beclin 1表达(3.62±2.69)显著低于对照组Beclin 1表达(10.32±4.12)(P<0.01)。腺肌病病灶组Beclin 1表达(0.27±0.52)显著低于在位腺肌病组及对照组(P<0.01)。在位腺肌病组HIF-1α表达(8.70±2.13)显著高于对照组HIF-1α表达(4.49±2.55)(P<0.01)。腺肌病病灶组HIF-1α表达(7.97±2.54)显著高于对照组(P<0.01),与在位腺肌病组之间无显著性差异。腺肌病组Beclin 1表达增殖期与分泌期之间无显著性差异,对照组Beclin 1表达增殖期与分泌期之间无显著性差异。增殖期Beclin 1表达腺肌病组与对照组间有显著性差异(P<0.01)。分泌期Beclin 1表达腺肌病组与对照组间有显著性差异(P<0.01)。腺肌病组HIF-1α表达增殖期与分泌期之间无显著性差异,对照组HIF-1α表达增殖期与分泌期之间有显著性差异(P<0.01)。增殖期HIF-1α表达腺肌病组显著高于对照组(P<0,01)。分泌期HIF-1α表达腺肌病组与对照组间无显著性差异。腺肌病病灶组Beclin 1表达显著低于腺肌病增殖期组、腺肌病分泌期组、对照增殖期组及对照分泌期组(P<0.01)。腺肌病病灶组Beclin 1表达显著低于HIF-1α表达(P<0.01)。腺肌病病灶组HIF-1α表达显著高于对照增殖期组(P<0.01),与腺肌病增殖期组、腺肌病分泌期组及对照分泌期组之间均无显著性差异。3.Beclin 1表达水平与血清CA125水平呈负相关(n=140,r2=0.232,P=0.000),与盆腔疼痛(VAS评分)亦呈负相关(n=140,r2=0.508,P=0.000)。4.HIF-1α表达水平与血清CA125水平呈正相关(n=140,r2=0.268,P=0.000)。HIF-1α表达水平与盆腔疼痛(VAS评分)亦呈正相关(n=140,r2=0.574,P=0.000)。5.Beclin 1表达水平与HIF-1α表达水平呈负相关(n=140,r2=0.201,P=0.000)。第三部分Beclin 1在子宫腺肌病在位内膜间质细胞中的表达及缺氧对其的影响目的：探讨Beclin 1在子宫腺肌病子宫内膜间质细胞的表达及缺氧对其的影响,并对缺氧培养24小时后Beclin 1表达水平与其增殖、迁移能力进行相关性分析。方法：对子宫内膜间质细胞进行原代培养并进行缺氧24小时处理,通过RT-PCR、蛋白免疫印迹技术、MTT及细胞迁移实验观察其增殖、迁移能力变化以及Beclin 1表达变化。结果：1.通过灰度分析,子宫腺肌病组Beclin 1 mRNA相对表达量为(0.72±0.19),明显低于对照组(1.17±0.44)(P<0.05)。子宫腺肌病组Beclin 1蛋白相对表达量为(0.20±0.03),明显低于对照组(0.71±0.12)(P<0.01)。2.MTT比色法发现,腺肌病常氧培养组OD值为(0.33±0.03),明显高于对照常氧培养组(0.24±0.04)(P<0.01),腺肌病缺氧培养组OD值为(0.46±0.03),明显高于腺肌病常氧培养组(0.33±0.03)(P<0.01),对照缺氧培养组OD值为(0.20±0.02),明显低于对照常氧培养组(0.24±0.04)(P<0.05)。3.细胞迁移实验发现,腺肌病常氧培养组细胞迁移距离为(0.31±0.02mm),明显高于对照常氧培养组(0.28±0.02mm)(P<0.01),腺肌病缺氧培养组细胞迁移距离为(0.37±0.02mm),明显高于腺肌病常氧培养组(0.31±0.02mm)(P<0.01),对照缺氧培养组细胞迁移距离为(0.14±0.02mm),明显低于对照常氧培养组(0.28±0.02mm)(P<0.01)。4.腺肌病缺氧组Beclin 1蛋白相对表达量为(0.30±0.01),明显低于腺肌病常氧组(0.49±0.01)(P<0.01)。腺肌病缺氧组Beclin 1蛋白相对表达量为(0.30±0.01),明显低于对照缺氧组(0.65±0.02)(P<0.01)。对照缺氧组Beclin 1蛋白相对表达量为(0.65±0.02)与对照常氧组(0.70±0.03)之间无统计学差异(P>0.05)。腺肌病常氧组Beclin 1蛋白相对表达量为(0.49±0.01),明显低于对照常氧组(0.70±0.03)(P<0.01)。5.通过Pearson相关分析发现,腺肌病在位内膜间质细胞缺氧培养24小时后Beclin 1蛋白表达水平与MTT所测的OD值,迁移距离呈明显的负相关关系(r2=0.564,0.469；P=0.004,0.042)。结论1. Beclin 1蛋白定位于子宫内膜腺细胞和间质细胞。子宫腺肌病在位内膜和病灶Beclin 1表达均下调,Beclin 1蛋白表达与血清CA125水平和盆腔疼痛呈负相关。Beclin 1可能在子宫腺肌病的发病和进展中发挥作用。2.HIF-1α蛋白定位于子宫内膜腺细胞和间质细胞,病灶以腺上皮表达为主。子宫腺肌病在位内膜和病灶HIF-1α表达均上调,HIF-1α表达水平与血清CA125水平及盆腔疼痛呈正相关。HIF-1α可能在子宫腺肌病的发病和进展中发挥作用。3. Beclin 1表达水平与HIF-1α表达水平呈负相关。HIF-1α表达越高、Beclin 1表达越低,可能预示着子宫腺肌病病情越严重。4.腺肌病在位内膜间质细胞增殖和迁移能力明显高于正常对照组。腺肌病在位内膜间质细胞缺氧培养24小时后增殖和迁移能力明显高于腺肌病常氧培养组。对照组在位内膜间质细胞缺氧培养24小时后增殖和迁移能力明显低于对照常氧培养组。缺氧使腺肌症在位内膜间质细胞拥有更强的增殖和迁移能力。5.腺肌病在位内膜间质细胞缺氧培养24小时后Beclin 1蛋白表达明显低于腺肌病常氧培养组。对照缺氧组Beclin 1蛋白表达与对照常氧组之间无统计学差异。6.腺肌病在位内膜间质细胞缺氧培养24小时后Beclin 1蛋白表达水平与MTT所测的OD值,迁移距离呈明显的负相关关系,缺氧诱导Beclin1变化可能在子宫腺肌病致病过程中发挥着作用。更多还原

【Abstract】 Adenomyosis is a nontumorous condition characterized by the presence of ectopic endometrium in the myometrium with hyperplasia of adjacent smooth muscle. This probably occurs by invagination of the basalis endometrium into the myometrium. The process of invagination and intramyometrial dissemination may be facilitated by the non-cyclic, anti-apoptotic activity of the basalis associated with relative hyper-oestrogenic states. Most cases of adenomyosis are discovered in multiparous women during the transitional years (40-50 years). It can result in debilitating pelvic pain (both cyclical and non-cyclical) and abnormal uterine bleeding. Magnetic resonance imaging establishes the diagnosis in cases of equivocal or nondiagnostic ultrasounds. Definite diagnosis and treatment of adenomyosis are obtained by hysterectomy. Vessel embolization, hormonal treatments (progestagens, continuous oral contraceptive pills, anti-estrogens and gonadotrophin-releasing hormone (GnRH) analogues) and high-intensity focused ultrasound can be tried in infertile women, however, the effect of these treatments is limited. Surgical extirpation has been the best therapeutic option for symptom relief so far. The etiology, pathogenesis and the better conservative treatment are still unknown to date.Autophagy, characterized by autophagic vacuoles accumulating, is considered as programmed cell death typeⅡwhich is known as autophagic cell death. Autophagy is involved not only in the balance between protein synthesis and degradation but also in the balance between cell survival and cell death. Beclin 1, a haploinsufficient tumor suppressor gene on chromosome 17q21 and a mammalian homolog of yeast Atg6/Vps30, is monoallelically deleted in up to 75% of ovarian cancers,50% of breast cancers and 40% of prostate cancers. Reduction of Beclin 1 is also observed in other cancers including human brain tumors and cervical carcinoma. Overexpression of Beclin 1 not only promotes nutrient deprivation-induced autophagy but also inhibits clonigenicity and tumorigenesis in human breast carcinoma cells. Structural studies display that Beclin 1 has a BH3-only domain, a central coiled-coil domain (CCD), and an evolutionarily conserved domain (ECD). The ECD of Beclin 1 is required for Vps34 binding, autophagy and tumor suppression. The BH3-only domain can interact with Bcl-2 and Bcl-XL. The Beclin 1 protein is an important convergence point of autophagy and apoptosis; it interacts with the anti-apoptotic and anti-autophagic Bcl-2 protein.Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. HIF-1 transcriptional activity is determined by regulated expression of the HIF-la subunit. HIF-1a is a transcriptional factor that regulates genes involved in response to hypoxia and promotes neoangiogenesis, which are considered essential for tumor growth and progression. Under normoxic conditions it is constitutively produced and degraded by the ubiquitin-proteasome system. But under hypoxic conditions it becomes stabilized.Hypoxia is associated with autophagy. Mitochondrial autophagy is induced by hypoxia, this process requires the HIF-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1. In cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.Researchers have launched a new area of febrile investigations on the autophagy-related gene Beclin 1. However, no study has previously shown the contribution of Beclin 1 to adenomyosis and the correlation between Beclin 1 and HIF-1a. This study investigated the expression of Beclin 1 in human adenomyosis, its association with clinical characteristics and its regulation in endometrial stromal cells by hypoxia. Cell cultures, immunohistochemical technique, RT-PCR, and western blotting were used in this study. The study was divided into three parts:1. Decreased expression of Beclin 1 in eutopic endometrium of women with adenomyosis and its association with clinical characteristics.2. Beclin 1, HIF-1αexpression in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.3. Beclin 1 expression in endometrial stromal cells from adenomyois and its regulation by hypoxia.Part one Decreased expression of Beclin 1 in eutopic endometrium of women with adenomyosis and its association with clinical characteristics.Objective:To investigate whether Beclin 1 expression is altered in eutopic endometrium of women with adenomyosis.Methods:We collected tissue samples from the eutopic endometria of 30 women with adenomyosis and 32 healthy women undergoing surgery for benign indications. Beclin 1 expression of eutopic endometrial tissues was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.Results:Beclin 1 mRNA expression level in tissue samples of women with adenomyosis (0.71±0.10) was significantly lower than that of control (0.93±0.10) (P<0.001). The average level of Beclin 1 protein expression of women with adenomyosis in tissue samples (0.82±0.22) was also significantly lower than that of control (1.22±0.36) (P<0.001). Beclin 1 protein expression in eutopic endometrial tissues was negatively correlated with serum CA125 (r=-0.307, P=0.015), and pelvic pain (r=-0.542, P=0.000).Part two Beclin 1, HIF-la expression in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.Objective:To investigate whether Beclin 1 and HIF-la were expressed in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.Methods: Beclin 1 and HIF-1αexpression were assessed by immunohistochemistry.Results:1. Beclin 1 immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells. HIF-1αimmunoreactive staining was present in the cytoplasm and nucleus of glandular epithelial and stromal cells in eutopic endometrium, mainly in the cytoplasm of glandular epithelial cells in adenomyotic foci. 2. Beclin 1 expression in eutopic endometrium of adenomyosis (3.62±2.69) was significantly lower than that in control (10.32±4.12)(P<0.01). Beclin 1 expression in adenomyotic foci (0.27±0.52) was significantly lower than that in eutopic endometrium of adenomyosis or in control (P<0.01). HIF-1αexpression in eutopic endometrium of adenomyosis (8.70±2.13) was significantly higher than that in control (4.49±2.55)(P<0.01). HIF-1αexpression in adenomyotic foci(7.97±2.54)was significantly higher than that in control(P<0.01), and not different from that in eutopic endometrium of adenomyosis. There were no differences of Beclin 1 expression between proliferative period and secretory period in adenomyosis or control. Beclin 1 expression of proliferative period or secretory period in adenomyosis was significantly lower than that in control(P<0.01). There were no differences of HIF-1αbetween proliferative period and secretory period in adenomyosis. There were differences of HIF-1αexpression between proliferative period and secretory period in control (P<0.01). HIF-1αexpression of proliferative period in adenomyosis was significantly higher than that in control(P<0.01). There were no differences of HIF-1αbetween adenomyosis and control in secretory period. Beclin 1 expression in adenomyotic foci was significantly lower than HIF-1αexpression(P<0.01).3. Beclin 1 expression was negatively correlated with serum CA125 (n=140, r2=0.232, P=0.000), and pelvic pain (n=140, r2=0.508, P=0.000). HIF-1αexpression was positively correlated with serum CA125 (n=140, r2 =0.268, P=0.000), and pelvic pain (n=140, r2=0.574, P=0.000). Beclin 1 expression was negatively correlated with HIF-1αexpression (n=140, r2=0.201, P=0.000).Part three Beclin 1 expression in endometrial stromal cells from adenomyois and its regulation by hypoxiaObjective:To investigate whether Beclin 1 expression is altered in endometrial stromal cells from adenomyosis and its regulation by hypoxia.Methods:Beclin 1 expression was assessed by RT-PCR and western blot analysis. The capability of proliferation and metastasis of endometrial stromal cells were assessed by MTT and migration assay.Results:1. Beclin 1 mRNA expression level in cultured stromal cells of women with adenomyosis (0.72±0.19) was significantly lower than that of without (1.17±0.44) (P<0.05). Beclin 1 protein expression of women with adenomyosis in cultured stromal cells (0.20±0.03) was significantly lower compared with that of control (0.71±0.12)(P<0.01).2. The growth rate by MTT assay and the migrated distance of endometrial stromal cells in adenomyosis (0.33±0.03,0.31±0.02mm, respectively) were significantly higher than those in the control group (0.24±0.04,0.28±0.02mm, respectively) (P<0.01). The growth rate by MTT assay and the migrated distance of endometrial stromal cells after 24h hypoxia in adenomyosis (0.46±0.03,0.37±0.02mm, respectively) were significantly higher than those under normoxia in adenomyosis (0.33±0.03,0.31±0.02mm, respectively) (P<0.01). The growth rate by MTT assay and the migrated distance of endometrial stromal cells after 24h hypoxia in control (0.20±0.02,0.14±0.02mm, respectively) were significantly lower than those under normoxia in control (0.24±0.04,0.28±0.02mm, respectively) (P<0.05).3. Beclin 1 protein expression of endometrial stromal cells after 24h hypoxia in adenomyosis (0.30±0.01) was significantly lower than that in adenomyosis under normoxia (0.49±0.01) (P<0.01)。Beclin 1 protein expression of endometrial stromal cells after 24h hypoxia in adenomyosis (0.30±0.01) was significantly lower than that in control under hypoxia (0.65±0.02) (P<0.01)。Beclin 1 protein expression of endometrial stromal cells from in adenomyosis (0.49±0.01) was significantly lower than that in control (0.70±0.03) (P<0.01)。There was no difference between the control group under hypoxia (0.65±0.02) and the control group under normoxia (0.70±0.03).4. In endometrial stromal cells after 24h hypoxia from adenomyosis, Beclin 1 protein level was negatively related with the cell proliferation and migration(r2=0.564,0.469;P=0.004,0.042). Conclusion:1. Beclin 1 immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells. Expression of Beclin 1 was decreased in eutopic endometrium and adenomyotic foci of women with adenomyosis, and the Beclin 1 expression was negatively correlated with the serum CA125 and pelvic pain, which might be related to the pathogenesis and progress of adenomyosis.2. HIF-1αimmunoreactive staining was present in the cytoplasm and nucleus of glandular epithelial and stromal cells in eutopic endometrium, mainly in the cytoplasm of glandular epithelial cells in adenomyotic foci. Expression of HIF-1αwas increased in eutopic endometrium and adenomyotic foci of women with adenomyosis. Its expression level was positively correlated with the serum CA125 and pelvic pain, which might be related to the pathogenesis and progress of adenomyosis.3. Beclin 1 expression was negatively correlated with HIF-1αexpression. If the expression of HIF-1αis higher and the expression of Beclin 1 is lower, the adenomyosis is more severe.4. The growth rate and the migrated distance of endometrial stromal cells in adenomyosis were significantly higher than those in control. The growth rate and the migrated distance of endometrial stromal cells after 24h hypoxia in adenomyosis were significantly higher than those under normoxia in adenomyosis. The endometrial stromal cells of adenomyosis had the greater capacity of proliferation and migration induced by hypoxia.5. Beclin 1 protein expression of endometrial stromal cells under hypoxia in adenomyosis was significantly lower than that under normoxia in adenomyosis。Beclin 1 protein expression of endometrial stromal cells under hypoxia in adenomyosis was significantly lower than that under hypoxia in control.6. In endometrial stromal cells after 24h hypoxia from adenomyosis, Beclin 1 protein level was negatively related with the cell proliferation and migration, hypoxia-induced change of Beclin 1 might be related to the pathogenesis and progress of adenomyosis.更多还原