水通道蛋白在子宫内膜异位症的表达及子宫内膜细胞迁徙中的作用
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摘要
研究背景
子宫内膜异位症是生育期女性的常见病,且近年来有上升趋势；本病在病理上呈良性形态学表现,但具有类似恶性肿瘤的特性：种植、侵袭及远处转移,是当今严重威胁妇女健康的妇科主要疾病之一。异位子宫内膜的来源至今尚未阐明,目前主要学说有：子宫内膜种植学说、淋巴及静脉播散学说、体腔上皮化生学说、诱导学说、遗传学说、免疫调节学说、其他因素(包括环境因素、血管生成、凋亡减少等)。其中种植学说是被绝大多是学者接受的,但是在位子宫内膜是如何种植到子宫腔以外的部位的目前尚不明确。子宫内膜异位症是一个有侵袭能力的良性疾病,侵袭有三部曲：迁徙、粘附和血管形成。在子宫内膜异位症中,关于在位内膜和异位内膜的粘附和血管形成的研究较多,目前尚无子宫内膜异位症迁徙方面的研究。
子宫内膜异位症的发生是一个不断进展的过程,如何预防疾病的复发已经成了一个国际性难题。这使得从疾病发生之初阻断异位内膜的种植尤为关键。因此,揭示子宫内膜种植过程中子宫内膜细胞迁徙的分子机制对防治子宫内膜异位症的发生具有重要的意义。
细胞迁徙是机体内重要的生理过程,它是由离子通道调控的,涉及到细胞肌动蛋白的动力和形态的快速改变、·细胞粘附位点的重新分布。细胞内存在一套完整、严密的机制以调控细胞的迁徙。细胞的迁徙可以分解成三个步骤：细胞的前沿伸出伪足,肌动蛋白网收缩,引导细胞迁徙的方向；未聚合的激动蛋白随伪足运动,形成新的肌动蛋白,伪足与底物结合产生新的粘附；尾部的胞体粘附分离,细胞向前移动；尾部的激动蛋白收缩,胞体向前移动。细胞迁徙发生的原因与细胞体内渗透压的改变有关。引起细胞体内渗透压的改变需要大量水分子的参与。目前关于细胞迁徙与子宫内膜异位症的发生的研究较少。
长期以来普遍认为,细胞内外的水分子是以简单的跨膜扩散方式透过脂质双分子层。1988年,Agre等在鉴定人类Rh血型抗原时,偶然在红细胞膜上发现了一种新的28ku跨膜蛋白。1991—-1992年,Agre等又先后完成了该蛋白分子克隆和功能鉴定,并证实该蛋白具有明确的水转运功能。随后,在许多动、植物及微生物中相继发现类似的专一性运输水的通道,统称为水通道蛋白(aquaporin,AQP)。研究表明AQP家族有着广泛的功能：促进水分子的快速被动运输；调节细胞内外渗透压的平衡；促进细胞迁徙；调节脂肪代谢；神经信号传导等。在女性生殖器官中的子宫、卵巢、输卵管部位,卵泡的排卵、月经的形成以及恶性肿瘤或具有恶性行为的良性妇科疾病的发生发展都涉及到液体的流动以及宫腔或卵泡腔容积的改变。
在女性的在位子宫内膜细胞上已经发现有AQP的表达,我院前期的研究发现,AQP1,2,8在在位子宫内膜上有表达,AQP5在卵巢癌上皮细胞上有表达；但迄今AQP在异位子宫内膜细胞上的研究尚未见相关报道。
由于本院前期的研究已经证明AQP1,2,8在正常妇女的子宫内膜上有表达,尚无水通道蛋白在子宫内膜异位症中的研究。本研究首先检测子宫内膜异位症患者的在位子宫内膜和异位子宫内膜的AQP1,2,5,8的表达情况,以探讨水通道蛋白在子宫内膜异位症的子宫内膜中的表达定位；结果发现,AQP1在在位内膜和异位内膜的微血管上有表达,AQP2,5,8在在位内膜和异位内膜的腺细胞上均有表达。AQP1、2、4、5、8是选择性运输水分子的水通道蛋白,由于本院对AQP1在正常子宫内膜微血管、子宫内膜增殖症以及子宫内膜癌中已有研究,文献报道AQP2、8主要是在分泌细胞上表达,本研究发现,AQP2、5、8在有分泌功能的子宫内膜腺上皮细胞上有表达,目前尚无AQP5在分泌细胞上的研究。本研究首先检测子宫内膜异位症患者的在位和异位内膜的AQP1,2,5,8的表达情况,以探讨AQP1,2,5,8在异位子宫内膜中的定位；然后以AQP5为切入点,应用RNA干扰技术沉默AQP5基因,观察干扰后子宫内膜腺细胞AQP5的表达及其干扰后细胞迁徙的改变；旨在阐明细胞迁徒与子宫内膜异位症发生的关系,并寻找能阻断子宫内膜迁徙的关键分子,为探索子宫内膜异位症的防治提供实验依据。
第一章
AQP1,2,5,8蛋白在子宫内膜异位症在位和异位内膜组织中的表达
研究目的
分别检测子宫内膜异位症患者的在位内膜和异位内膜的AQP1,2,5,8的表达水平,初步探讨水通道蛋白的表达与子宫内膜异位症的发生、发展的关系。
材料和方法
选取2004年10月至2007年9月在浙江大学医学院附属妇产科医院病理科存档的,因子宫内膜异位症进行半根治手术或术中诊刮的内膜,获得符合条件的病例共70例。根据纳入标准获得70例患者的异位内膜及在位内膜的石蜡标本。实验组为异位子宫内膜,对照组为同一例患者的在位内膜,通过免疫组织化学的方法检测AQP1,2,5,8蛋白的表达情况。
结果
1.AQP1在在位内膜和异位内膜的微血管中有丰富表达,在间质细胞和腺细胞中无表达；在在位子宫内膜组中,AQP2,5,8蛋白的表达主要位于子宫内膜的腺上皮和腔上皮细胞；AQP2在间质中无表达,AQP8在间质中有少许表达,AQP5在间质中有较弱的表达；在异位子宫内膜中,AQP2,5,8的表达主要在腺上皮和腔上皮细胞,在间质中的表达略高于在位内膜。2.在两组中,AQP1,2,5,8的表达与月经周期有关,在增生早期的表达较弱,增生晚期和分泌期的表达较强。3.光密度分析AQP1在分泌期的异位内膜的表达高于在位内膜；半定量分析AQP2,5,8在在位内膜的表达显著高于异位内膜上的表达(P=<0.001,P=0.004,P<0.001)；4.AQP1,2,5,8在在位内膜和异位内膜的表达在增生期和分泌期无显著性差异。
结论AQP1,2,5,8在子宫内膜上的过表达可能参与子宫内膜异位症的发生。
第二章
干扰AQP5基因对子宫内膜腺细胞迁徙的影响
研究目的
探讨AQP5基因对子宫内膜腺细胞的迁徙的影响,从而明确迁徙相关水通道蛋白基因对子宫内膜腺细胞迁徙的影响。
材料和方法
通过RNA干扰(RNAi)技术沉默子宫内膜癌腺细胞株Ishikawa中AQP5基因,Realtime-PCR检测干扰后mRNA水平的抑制,免疫印迹检测干扰后腺细胞中AQP5蛋白表达的改变；细胞穿孔实验检查干扰后腺细胞的迁徙能力的改变。
结果
1.AQP5 RNA干扰后,干扰组子宫内膜腺细胞AQP5 mRNA及蛋白表达水平明显降低,有统计学差异(P分别为0.03,0.007),而阴性对照组与空白对照组之间的mRNA及蛋白表达水平改变无统计学差异(P>0.05)。2.AQP5 RNA干扰后,RNA干扰组子宫内膜腺细胞的细胞迁徙数目显著下降(P<0.05)；阴性对照组与空白对照组间上述细胞穿过数的改变均无统计学差异。
结论
1.通过RNA干扰能成功沉默子宫内膜腺细胞中的AQP5基因；2.下调AQP5基因的表达可抑制子宫内膜腺细胞的迁徙,推测AQP5基因可影响子宫内膜腺细胞的迁徙。
Background
Endometriosis is a common disease of women in reproductive period, it shows up ward trend in recent years; this is a benign disease in pathological morphology of performance. However, it has similar characteristics with malignant tumors:planting, invasive and distant metastasis, today it has been one of the major in gynecological disease and a serious threat to women's health. The source of ectopic endometrium has not yet been clarified, at present the main theories are:cultivation of endometrial theory, lymphatic and venous spread theory, body cavity metaplasia theory, induction theory, genetics, immune regulation theory and other factors (including environmental factors, angiogenesis, apoptosis reduction, etc.). Cultivation of endometrial theory was accepted by most scholars, but how the eutopic endometrium grows into the uterine cavity area outside the present is not clear.
Endometriosis is a benign disease with invasion capability, invasion are often accompanied by Trilogy:migration, adhesion and angiogenesis. In endometriosis, there are much studies about the eutopic endometrium and ectopic endometrium on the adhesion and angiogenesis. There is no endometriosis migration research. The occurrence process of endometriosis is a continuous progress, how to prevent recurrence of the disease has become an international problem, which makes it particularly critical blocking the disease occurs from the beginning of the planting ectopic endometrium. Thus, reveal the molecular mechanism of the growing process of endometrial cells in movement and control the occurrence of endometriosis is of great significance.
As we all know, cell migration is an important physiological process of the body, which is regulated by ion channels, involved in actin dynamics and morphology of the rapid changes in cell and adhesion sites to re-distribution. Cells exist in a complete set of strict mechanisms to regulate cell migration. Cell migration can be decomposed into three steps:Cells in the forefront of extending pseudopodia, actin network contraction, and guide the direction of cell migration; actin dispolymerization with pseudopodia movement, the formation of new actin pseudopod with the election of a new substrate-binding adhesion; tail separation of the cell body adhesion and cell to move forward; tail actin contraction, cell body to move forward. Cell migration occurs because cells in vivo osmotic pressure changes. Changes in osmotic pressure caused by cells in vivo require the participation of a large number of water molecules. At present there is less research on the cell migration and the occurrence of endometriosis
For a long time, it is widely believed that water molecules inside and outside cells in simple ways, through the proliferation of lipid membrane bilayer. In 1988, Agre, etc. occasionally found a new 28ku transmembrane protein in the red blood cell membrane when identified the human Rh blood group antigen.1991-1992, Agre, etc. have completed the cloning and functional identification of proteins and confirmed that the protein has a clear water transport function. Subsequently, it was found a similar specificity in many animals, plants and microorganisms in the transport of water channels, known collectively as the water channel protein (Aquaporin, AQP). Studies have shown that AQP family has a wide range of functions:to promote the rapid passive transport of water molecules; regulate cell osmotic pressure inside and outside of the balance; promote cell migration; regulation of fat metabolism; neural signal transduction. In the female reproductive organs, including the uterus, ovaries, fallopian tubes parts follicle ovulation, menstruation, as well as the formation of the malignant behavior of cancer or the occurrence and development with benign gynecological diseases are related to fluid flow, and intrauterine or follicular cavity volume changes. AQP is a selective water channel water molecules through the pore, broad participation in reproductive organs of the physiological and pathological mechanisms occurring. AQP is a group of selectively transport water molecules across cell membrane of the small and complete glycoprotein, molecular weight of 25-34kD, some AQP subunit can also transport glycerol. AQP's first grade structure across the membrane six times a single peptide chain and its amino and carboxyl terminal are located in the cell, including three cell outer ring and two cell inner ring, the highest homology is located in ring asparagine-feeding acid-alanine (Asn-Pro-Ala, NPA) unit, NPA is a characteristic shared by members of the AQP family structures. AQP's tertiary structure was an hourglass pattern, allowing water molecules (H2O) free passage, but does not allow the water quality sub (H3O) through. AQPs are widely distributed in body tissue, particularly with the absorption of fluid secretion on the epithelial cells and endothelial cells in the content of their subsequent participation in water secretion, absorption and cellular water balance inside and outside. It has now been found in mammals,13 types of aquaporins (AQP0～AQP12), according to the infiltration of specific AQP, they will be divided into two categories, general and specific amino acid sequence match. Class 1 are permeable only to water, including AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP11, AQP12; the first two categories, including AQP3, AQP7, AQP8, AQP9, and AQP10, except transfer to water, but also for other small solutes are permeable, especially glycerol. Eutopic endometrium in women's cells have been found to have AQP expression, our hospital's pre-study found that AQP1,2,8 expressed in the eutopic endometrium, AQP5 expressed in epithelial ovarian cancer cells; but the study of AQP in the ectopic endometrial cells has yet to see the relevant reports.
In our hospital, other's early studies have shown that AQP1,2,5,8 expressed in the normal endometrium of healthy women. There is no aquaporin study in endometriosis. This study first detected in patients with endometriosis in eutopic endometrium and ectopic endometrial, explore AQP1,2,5,8 expression in endometriosis, observe AQP1,2,5,8 localization of endometrium; The results showed that, AQP1 located in microvascular in eutopic and ectopic endometrial; AQP2,5,8 located in endometrial glandular cells in eutopic and ectopic endometrium. AQP1,2,4,5,8 selective transport of water molecules of water channel proteins, This hospital has studied AQP1 in normal endometrial capillaries, endometrial hyperplasia and endometrial carcinoma. It has been reported in the literature that AQP2,8 mainly expressed in the secretory cells, at present there is no AQP5 study in secretory cells. This study found that AQP2,5,8 expressed in endometrial glandular epithelial cells, one type of the secretory epithelial cells. To explore AQPs function in the occurrence of endometriosis, design AQP5 as the entry point, apply RNA interference gene silencing AQP5 gene, observe expression of AQP5 in endometrial glandular cells and cell migration changes after RNAi AQP5. Seeks to clarify relationship between cell migration and endometriosis and searching for a key to block the migration of endometrial molecules, in order to provide experimental basis for exploring the prevention and treatment of endometriosis.
PartⅠ
AQP1,2,5,8 Proteins Expression in Eutopic and Ectopic Endometrial Tissues
Objective
To detect AQP1,2,5,8 expression level in eutopic and ectopic endometrium in patients with endometriosis, preliminary study the relationship between water channel protein(Aquaporin AQP) and the occurrence of endometriosis.
Materials and methods
The specimens (n=70) were obtained from the Pathological Department of Women's Hospital, School of Medicine, Zhejiang University from October 2004 to June 2007, and comprised 70 eutopic endometria,70 ectopic endometrial form endometriomas. Through the immunohistochemical detection of AQP1,2,5,8 protein expression.
Results
1. AQP1 was mainly abundantly located in microvascular in the eutopic and ectopic endometrial cells. Optical dentisy analysied there was statistical difference of AQP1 expressions between ectopic and eutopic groups(P<0.01). In samples from ectopic group, the AQP1 staining was different in proliferative and secretory phases(P<0.05), but not in eutopic endometria(P>0.05). The AQP1 expression was significantly higher in ectopic endometria than eutopic endometria in proliferativ phase(P<0.05) but not in secretory phase(P>0.05). In eutopic endometrium group, aquaporins 2,5, and 8 were mainly located in luminal and glandular epithelia. The frequency of positive immunostaining for aquaporins 2,5, and 8 decreased in ectopic compared with eutopic endometrial(p<0.001,p=0.004,p<0.001). Aquaporin 2,5, and 8 were found at a low frequency in the endometria in early proliferative phases but at a higher frequency in late proliferative and secretory phases. There were no significant differences in the menstrual cycle of the proliferative phase and secretory phase in the two groups.
Conclusions
Over expression of aquaporin 1,2,5,8 in endometrium involved in occurence of endometriosis.
PartⅡ
Role of RNAi AQP5 on Cell Migration in Endometrial Glandular cell
Objective
To explore AQP5 gene impact of migration in endometrial glandular cells, clarify migration-related water channel protein gene on the endometrium grown in vitro.
Materials and methods
Through RNA interference (RNAi) technology, silent endometrial cancer glandular cell line in the AQP5 gene, Realtime-PCR test mRNA level after RNAi AQP5. Western blot detection AQP5 protein expression changes of glandular cell line after interfence; endometrial glandular cells migration ability changes after siRNA AQP5 through cell perforation experiment examines(Transwell).
Results
1. After RNAi AQP5 gene, in interference group, AQP5 mRNA and protein expression levels of endometrial glandular cells reduced appearently, there were statistically significant (P=0.03 and 0.007 respectively), while the negative control group and blank control group, the mRNA and protein expression were no statistical difference in the level of change.2. After AQP5 RNA interference, in interference group endometrial glandular cells migration number decreased significantly compared with the negative control group and blank control group (P<0.05)
Conclusions
1. RNAi is able to silence AQP5 gene in endometrial glandular cells.
2. Down-regulated AQP5 expressions may inhibit the migration of endometrial glandular cells.
子宫内膜异位症是生育期女性的常见病,且近年来有上升趋势；本病在病理上呈良性形态学表现,但具有类似恶性肿瘤的特性：种植、侵袭及远处转移,是当今严重威胁妇女健康的妇科主要疾病之一。异位子宫内膜的来源至今尚未阐明,目前主要学说有：子宫内膜种植学说、淋巴及静脉播散学说、体腔上皮化生学说、诱导学说、遗传学说、免疫调节学说、其他因素(包括环境因素、血管生成、凋亡减少等)。其中种植学说是被绝大多是学者接受的,但是在位子宫内膜是如何种植到子宫腔以外的部位的目前尚不明确。子宫内膜异位症是一个有侵袭能力的良性疾病,侵袭有三部曲：迁徙、粘附和血管形成。在子宫内膜异位症中,关于在位内膜和异位内膜的粘附和血管形成的研究较多,目前尚无子宫内膜异位症迁徙方面的研究。
子宫内膜异位症的发生是一个不断进展的过程,如何预防疾病的复发已经成了一个国际性难题。这使得从疾病发生之初阻断异位内膜的种植尤为关键。因此,揭示子宫内膜种植过程中子宫内膜细胞迁徙的分子机制对防治子宫内膜异位症的发生具有重要的意义。
细胞迁徙是机体内重要的生理过程,它是由离子通道调控的,涉及到细胞肌动蛋白的动力和形态的快速改变、·细胞粘附位点的重新分布。细胞内存在一套完整、严密的机制以调控细胞的迁徙。细胞的迁徙可以分解成三个步骤：细胞的前沿伸出伪足,肌动蛋白网收缩,引导细胞迁徙的方向；未聚合的激动蛋白随伪足运动,形成新的肌动蛋白,伪足与底物结合产生新的粘附；尾部的胞体粘附分离,细胞向前移动；尾部的激动蛋白收缩,胞体向前移动。细胞迁徙发生的原因与细胞体内渗透压的改变有关。引起细胞体内渗透压的改变需要大量水分子的参与。目前关于细胞迁徙与子宫内膜异位症的发生的研究较少。
长期以来普遍认为,细胞内外的水分子是以简单的跨膜扩散方式透过脂质双分子层。1988年,Agre等在鉴定人类Rh血型抗原时,偶然在红细胞膜上发现了一种新的28ku跨膜蛋白。1991—-1992年,Agre等又先后完成了该蛋白分子克隆和功能鉴定,并证实该蛋白具有明确的水转运功能。随后,在许多动、植物及微生物中相继发现类似的专一性运输水的通道,统称为水通道蛋白(aquaporin,AQP)。研究表明AQP家族有着广泛的功能：促进水分子的快速被动运输；调节细胞内外渗透压的平衡；促进细胞迁徙；调节脂肪代谢；神经信号传导等。在女性生殖器官中的子宫、卵巢、输卵管部位,卵泡的排卵、月经的形成以及恶性肿瘤或具有恶性行为的良性妇科疾病的发生发展都涉及到液体的流动以及宫腔或卵泡腔容积的改变。
在女性的在位子宫内膜细胞上已经发现有AQP的表达,我院前期的研究发现,AQP1,2,8在在位子宫内膜上有表达,AQP5在卵巢癌上皮细胞上有表达；但迄今AQP在异位子宫内膜细胞上的研究尚未见相关报道。
由于本院前期的研究已经证明AQP1,2,8在正常妇女的子宫内膜上有表达,尚无水通道蛋白在子宫内膜异位症中的研究。本研究首先检测子宫内膜异位症患者的在位子宫内膜和异位子宫内膜的AQP1,2,5,8的表达情况,以探讨水通道蛋白在子宫内膜异位症的子宫内膜中的表达定位；结果发现,AQP1在在位内膜和异位内膜的微血管上有表达,AQP2,5,8在在位内膜和异位内膜的腺细胞上均有表达。AQP1、2、4、5、8是选择性运输水分子的水通道蛋白,由于本院对AQP1在正常子宫内膜微血管、子宫内膜增殖症以及子宫内膜癌中已有研究,文献报道AQP2、8主要是在分泌细胞上表达,本研究发现,AQP2、5、8在有分泌功能的子宫内膜腺上皮细胞上有表达,目前尚无AQP5在分泌细胞上的研究。本研究首先检测子宫内膜异位症患者的在位和异位内膜的AQP1,2,5,8的表达情况,以探讨AQP1,2,5,8在异位子宫内膜中的定位；然后以AQP5为切入点,应用RNA干扰技术沉默AQP5基因,观察干扰后子宫内膜腺细胞AQP5的表达及其干扰后细胞迁徙的改变；旨在阐明细胞迁徒与子宫内膜异位症发生的关系,并寻找能阻断子宫内膜迁徙的关键分子,为探索子宫内膜异位症的防治提供实验依据。
第一章
AQP1,2,5,8蛋白在子宫内膜异位症在位和异位内膜组织中的表达
研究目的
分别检测子宫内膜异位症患者的在位内膜和异位内膜的AQP1,2,5,8的表达水平,初步探讨水通道蛋白的表达与子宫内膜异位症的发生、发展的关系。
材料和方法
选取2004年10月至2007年9月在浙江大学医学院附属妇产科医院病理科存档的,因子宫内膜异位症进行半根治手术或术中诊刮的内膜,获得符合条件的病例共70例。根据纳入标准获得70例患者的异位内膜及在位内膜的石蜡标本。实验组为异位子宫内膜,对照组为同一例患者的在位内膜,通过免疫组织化学的方法检测AQP1,2,5,8蛋白的表达情况。
结果
1.AQP1在在位内膜和异位内膜的微血管中有丰富表达,在间质细胞和腺细胞中无表达；在在位子宫内膜组中,AQP2,5,8蛋白的表达主要位于子宫内膜的腺上皮和腔上皮细胞；AQP2在间质中无表达,AQP8在间质中有少许表达,AQP5在间质中有较弱的表达；在异位子宫内膜中,AQP2,5,8的表达主要在腺上皮和腔上皮细胞,在间质中的表达略高于在位内膜。2.在两组中,AQP1,2,5,8的表达与月经周期有关,在增生早期的表达较弱,增生晚期和分泌期的表达较强。3.光密度分析AQP1在分泌期的异位内膜的表达高于在位内膜；半定量分析AQP2,5,8在在位内膜的表达显著高于异位内膜上的表达(P=<0.001,P=0.004,P<0.001)；4.AQP1,2,5,8在在位内膜和异位内膜的表达在增生期和分泌期无显著性差异。
结论AQP1,2,5,8在子宫内膜上的过表达可能参与子宫内膜异位症的发生。
第二章
干扰AQP5基因对子宫内膜腺细胞迁徙的影响
研究目的
探讨AQP5基因对子宫内膜腺细胞的迁徙的影响,从而明确迁徙相关水通道蛋白基因对子宫内膜腺细胞迁徙的影响。
材料和方法
通过RNA干扰(RNAi)技术沉默子宫内膜癌腺细胞株Ishikawa中AQP5基因,Realtime-PCR检测干扰后mRNA水平的抑制,免疫印迹检测干扰后腺细胞中AQP5蛋白表达的改变；细胞穿孔实验检查干扰后腺细胞的迁徙能力的改变。
结果
1.AQP5 RNA干扰后,干扰组子宫内膜腺细胞AQP5 mRNA及蛋白表达水平明显降低,有统计学差异(P分别为0.03,0.007),而阴性对照组与空白对照组之间的mRNA及蛋白表达水平改变无统计学差异(P>0.05)。2.AQP5 RNA干扰后,RNA干扰组子宫内膜腺细胞的细胞迁徙数目显著下降(P<0.05)；阴性对照组与空白对照组间上述细胞穿过数的改变均无统计学差异。
结论
1.通过RNA干扰能成功沉默子宫内膜腺细胞中的AQP5基因；2.下调AQP5基因的表达可抑制子宫内膜腺细胞的迁徙,推测AQP5基因可影响子宫内膜腺细胞的迁徙。
Background
Endometriosis is a common disease of women in reproductive period, it shows up ward trend in recent years; this is a benign disease in pathological morphology of performance. However, it has similar characteristics with malignant tumors:planting, invasive and distant metastasis, today it has been one of the major in gynecological disease and a serious threat to women's health. The source of ectopic endometrium has not yet been clarified, at present the main theories are:cultivation of endometrial theory, lymphatic and venous spread theory, body cavity metaplasia theory, induction theory, genetics, immune regulation theory and other factors (including environmental factors, angiogenesis, apoptosis reduction, etc.). Cultivation of endometrial theory was accepted by most scholars, but how the eutopic endometrium grows into the uterine cavity area outside the present is not clear.
Endometriosis is a benign disease with invasion capability, invasion are often accompanied by Trilogy:migration, adhesion and angiogenesis. In endometriosis, there are much studies about the eutopic endometrium and ectopic endometrium on the adhesion and angiogenesis. There is no endometriosis migration research. The occurrence process of endometriosis is a continuous progress, how to prevent recurrence of the disease has become an international problem, which makes it particularly critical blocking the disease occurs from the beginning of the planting ectopic endometrium. Thus, reveal the molecular mechanism of the growing process of endometrial cells in movement and control the occurrence of endometriosis is of great significance.
As we all know, cell migration is an important physiological process of the body, which is regulated by ion channels, involved in actin dynamics and morphology of the rapid changes in cell and adhesion sites to re-distribution. Cells exist in a complete set of strict mechanisms to regulate cell migration. Cell migration can be decomposed into three steps:Cells in the forefront of extending pseudopodia, actin network contraction, and guide the direction of cell migration; actin dispolymerization with pseudopodia movement, the formation of new actin pseudopod with the election of a new substrate-binding adhesion; tail separation of the cell body adhesion and cell to move forward; tail actin contraction, cell body to move forward. Cell migration occurs because cells in vivo osmotic pressure changes. Changes in osmotic pressure caused by cells in vivo require the participation of a large number of water molecules. At present there is less research on the cell migration and the occurrence of endometriosis
For a long time, it is widely believed that water molecules inside and outside cells in simple ways, through the proliferation of lipid membrane bilayer. In 1988, Agre, etc. occasionally found a new 28ku transmembrane protein in the red blood cell membrane when identified the human Rh blood group antigen.1991-1992, Agre, etc. have completed the cloning and functional identification of proteins and confirmed that the protein has a clear water transport function. Subsequently, it was found a similar specificity in many animals, plants and microorganisms in the transport of water channels, known collectively as the water channel protein (Aquaporin, AQP). Studies have shown that AQP family has a wide range of functions:to promote the rapid passive transport of water molecules; regulate cell osmotic pressure inside and outside of the balance; promote cell migration; regulation of fat metabolism; neural signal transduction. In the female reproductive organs, including the uterus, ovaries, fallopian tubes parts follicle ovulation, menstruation, as well as the formation of the malignant behavior of cancer or the occurrence and development with benign gynecological diseases are related to fluid flow, and intrauterine or follicular cavity volume changes. AQP is a selective water channel water molecules through the pore, broad participation in reproductive organs of the physiological and pathological mechanisms occurring. AQP is a group of selectively transport water molecules across cell membrane of the small and complete glycoprotein, molecular weight of 25-34kD, some AQP subunit can also transport glycerol. AQP's first grade structure across the membrane six times a single peptide chain and its amino and carboxyl terminal are located in the cell, including three cell outer ring and two cell inner ring, the highest homology is located in ring asparagine-feeding acid-alanine (Asn-Pro-Ala, NPA) unit, NPA is a characteristic shared by members of the AQP family structures. AQP's tertiary structure was an hourglass pattern, allowing water molecules (H2O) free passage, but does not allow the water quality sub (H3O) through. AQPs are widely distributed in body tissue, particularly with the absorption of fluid secretion on the epithelial cells and endothelial cells in the content of their subsequent participation in water secretion, absorption and cellular water balance inside and outside. It has now been found in mammals,13 types of aquaporins (AQP0～AQP12), according to the infiltration of specific AQP, they will be divided into two categories, general and specific amino acid sequence match. Class 1 are permeable only to water, including AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP11, AQP12; the first two categories, including AQP3, AQP7, AQP8, AQP9, and AQP10, except transfer to water, but also for other small solutes are permeable, especially glycerol. Eutopic endometrium in women's cells have been found to have AQP expression, our hospital's pre-study found that AQP1,2,8 expressed in the eutopic endometrium, AQP5 expressed in epithelial ovarian cancer cells; but the study of AQP in the ectopic endometrial cells has yet to see the relevant reports.
In our hospital, other's early studies have shown that AQP1,2,5,8 expressed in the normal endometrium of healthy women. There is no aquaporin study in endometriosis. This study first detected in patients with endometriosis in eutopic endometrium and ectopic endometrial, explore AQP1,2,5,8 expression in endometriosis, observe AQP1,2,5,8 localization of endometrium; The results showed that, AQP1 located in microvascular in eutopic and ectopic endometrial; AQP2,5,8 located in endometrial glandular cells in eutopic and ectopic endometrium. AQP1,2,4,5,8 selective transport of water molecules of water channel proteins, This hospital has studied AQP1 in normal endometrial capillaries, endometrial hyperplasia and endometrial carcinoma. It has been reported in the literature that AQP2,8 mainly expressed in the secretory cells, at present there is no AQP5 study in secretory cells. This study found that AQP2,5,8 expressed in endometrial glandular epithelial cells, one type of the secretory epithelial cells. To explore AQPs function in the occurrence of endometriosis, design AQP5 as the entry point, apply RNA interference gene silencing AQP5 gene, observe expression of AQP5 in endometrial glandular cells and cell migration changes after RNAi AQP5. Seeks to clarify relationship between cell migration and endometriosis and searching for a key to block the migration of endometrial molecules, in order to provide experimental basis for exploring the prevention and treatment of endometriosis.
PartⅠ
AQP1,2,5,8 Proteins Expression in Eutopic and Ectopic Endometrial Tissues
Objective
To detect AQP1,2,5,8 expression level in eutopic and ectopic endometrium in patients with endometriosis, preliminary study the relationship between water channel protein(Aquaporin AQP) and the occurrence of endometriosis.
Materials and methods
The specimens (n=70) were obtained from the Pathological Department of Women's Hospital, School of Medicine, Zhejiang University from October 2004 to June 2007, and comprised 70 eutopic endometria,70 ectopic endometrial form endometriomas. Through the immunohistochemical detection of AQP1,2,5,8 protein expression.
Results
1. AQP1 was mainly abundantly located in microvascular in the eutopic and ectopic endometrial cells. Optical dentisy analysied there was statistical difference of AQP1 expressions between ectopic and eutopic groups(P<0.01). In samples from ectopic group, the AQP1 staining was different in proliferative and secretory phases(P<0.05), but not in eutopic endometria(P>0.05). The AQP1 expression was significantly higher in ectopic endometria than eutopic endometria in proliferativ phase(P<0.05) but not in secretory phase(P>0.05). In eutopic endometrium group, aquaporins 2,5, and 8 were mainly located in luminal and glandular epithelia. The frequency of positive immunostaining for aquaporins 2,5, and 8 decreased in ectopic compared with eutopic endometrial(p<0.001,p=0.004,p<0.001). Aquaporin 2,5, and 8 were found at a low frequency in the endometria in early proliferative phases but at a higher frequency in late proliferative and secretory phases. There were no significant differences in the menstrual cycle of the proliferative phase and secretory phase in the two groups.
Conclusions
Over expression of aquaporin 1,2,5,8 in endometrium involved in occurence of endometriosis.
PartⅡ
Role of RNAi AQP5 on Cell Migration in Endometrial Glandular cell
Objective
To explore AQP5 gene impact of migration in endometrial glandular cells, clarify migration-related water channel protein gene on the endometrium grown in vitro.
Materials and methods
Through RNA interference (RNAi) technology, silent endometrial cancer glandular cell line in the AQP5 gene, Realtime-PCR test mRNA level after RNAi AQP5. Western blot detection AQP5 protein expression changes of glandular cell line after interfence; endometrial glandular cells migration ability changes after siRNA AQP5 through cell perforation experiment examines(Transwell).
Results
1. After RNAi AQP5 gene, in interference group, AQP5 mRNA and protein expression levels of endometrial glandular cells reduced appearently, there were statistically significant (P=0.03 and 0.007 respectively), while the negative control group and blank control group, the mRNA and protein expression were no statistical difference in the level of change.2. After AQP5 RNA interference, in interference group endometrial glandular cells migration number decreased significantly compared with the negative control group and blank control group (P<0.05)
Conclusions
1. RNAi is able to silence AQP5 gene in endometrial glandular cells.
2. Down-regulated AQP5 expressions may inhibit the migration of endometrial glandular cells.
引文
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[18]Yang JH, Shi YF, Cheng Q, Deng L. Expression and localization of aquaporin-5 in the epithelial ovarian tumors. Gynecol Oncol. 2006; 100(2):294-9.
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[20]Yang JH, Shi YF, Chen XD, Qi WJ. The influence of aquaporin-1 and microvessel density on ovarian carcinogenesis and ascites formation, Int J Gynecol Cancer. 2006;16 Suppl 1:400-5.
[21]Ji C, Cao C, Lu S, Kivlin R, Amaral A, Kouttab N, Yang H, Chu W, Bi Z, Di W, Wan Y. Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells.Cancer Chemother Pharmacol.2008; 62(5):857-65
[22]Papadopoulos MC, Saadoun S, Verkman AS. Aquaporins and cell migration.Pflugers Arch.2008; 456(4):693-700.
[23]Samira Saadoun2, Marios C. Papadopoulos2, Mariko Hara-Chikuma and A. S. Verkman. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature.2005; 434:786-792
[24]Verkman AS, Hara-Chikuma M, Papadopoulos MC. Aquaporins-new players in cancer biology. J Mol Med.2008; 86(5):523-9.
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[1]S. Verkman More than just water channels:unexpected cellular roles of aquaporins. Journal of Cell Science.2005; 118:3225-3232.
[2]Lindsay LA, Murphy CR Redistribution of aquaporins 1 and 5 in the rat uterus is dependent on progesterone:a study with light and electron microscopy. Reproduction.2006; 131(2):369-78.
[3]Kobayashi M, Takahashi E, Miyagawa S, Watanabe H, Iguchi T. Chromatin immunoprecipitation-mediated target identification proved aquaporin 5 is regulated directly by estrogen in the uterus. Genes Cells.2006;11(10):1133-43.
[4]Hildenbrand A, Lalitkumar L, Nielsen S, Gemzell-Danielsson K, Stavreus-Evers A. Expression of aquaporin 2 in human endometrium. Fertil Steril. 2006; 86(5):1452-8.
[5]He RH, Sheng JZ, Luo Q, Jin F, Wang B, Qian YL, Zhou CY, Sheng X, Huang HF. Aquaporin-2 expression in human endometrium correlates with serum ovarian steroid hormones. Life Sci.2006; 79(5):423-9.
[6]Lindsay LA, Murphy CR Redistribution of aquaporins 1 and 5 in the rat uterus is dependent on progesterone:a study with light and electron microscopy. Reproduction.2006;131(2):369-78.
[7]Lindsay LA, Murphy CR. Aquaporin-1 increases in the rat myometrium during early pregnancy. J Mol Histol.2004;35(1):75-9.
[8]Liu H, Wintour EM. Aquaporins in development-a review. Reprod Biol Endocrinol.2005; 3(18):1-10.
[9]Mann SE, Ricke EA, Torres EA, Taylor RN A novel model of polyhydramnios: amniotic fluid volume is increased in aquaporin 1 knockout mice. Am J Obstet Gynecol. 2005; 192(6):2041-4; discussion 2044-6.
[10]Sun CC, Feng C, Zhou CY, Huang HF. Expression profile of aquaporin 1 in patients with menorrhagia. Zhejiang Da Xue Bao Yi Xue Ban.2007;36(5):433-8.
[11]Mobasheri A, Wray S, Marples D. Distribution of AQP2 and AQP3 water channels in human tissue microarrays. J Mol Histol.2005; 36(1-2):1-14.
[12]Sun XL, Ding JH, Fan Y, Zhang J, Gao L, Hu G. Aquaporin 4 regulates the effects of ovarian hormones on monoamine neurotransmission. Biochem Biophys Res Commun.2007;353(2):457-62.
[13]Powers RW, Chen L, Russell PT, Larsen WJ. Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am J Physiol.1995; 269:E290-E298.
[14]Hess KA, Chen L, Larsen WJ. The ovarian blood follicle barrier is both charge-and size-selective in mice. Biol Reprod.1998; 58:705-711.
[15]McConnell NA, Yunus RS, Gross SA, Bost KL, Clemens MG, Hughes FM JR. Water permeability of an ovarian antral follicle is predominantly transcellular and mediated by aquaporins. Endocrinology.2002; 143(8):2905-2912.
[16]De Wilde J, Wilting SM, Meijer CJ, van de Wiel MA, Ylstra B, Snijders PJ, Steenbergen RD. Gene expression profiling to identify markers associated with deregulated hTERT in HPV-transformed keratinocytes and cervical cancer.Int J Cancer.2008 Feb 15; 122(4):877-88.
[17]Pan H, Sun CC, Zhou CY, Huang HF. Expression of aquaporin-1 in normal, hyperplasic, and carcinomatous endometria.Int J Gynaecol Obstet.2008; 101(3):239-44.
[18]Yang JH, Shi YF, Cheng Q, Deng L. Expression and localization of aquaporin-5 in the epithelial ovarian tumors. Gynecol Oncol. 2006; 100(2):294-9.
[19]Yang JH, Shi YF, Cheng Q, Deng L. Expression andlocalization of aquaporin-5 in the epithelial ovarian tumors. Gynecol Oncol.2006; 100:294-299.
[20]Yang JH, Shi YF, Chen XD, Qi WJ. The influence of aquaporin-1 and microvessel density on ovarian carcinogenesis and ascites formation, Int J Gynecol Cancer. 2006;16 Suppl 1:400-5.
[21]Ji C, Cao C, Lu S, Kivlin R, Amaral A, Kouttab N, Yang H, Chu W, Bi Z, Di W, Wan Y. Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells.Cancer Chemother Pharmacol.2008; 62(5):857-65
[22]Papadopoulos MC, Saadoun S, Verkman AS. Aquaporins and cell migration.Pflugers Arch.2008; 456(4):693-700.
[23]Samira Saadoun2, Marios C. Papadopoulos2, Mariko Hara-Chikuma and A. S. Verkman. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature.2005; 434:786-792
[24]Verkman AS, Hara-Chikuma M, Papadopoulos MC. Aquaporins-new players in cancer biology. J Mol Med.2008; 86(5):523-9.
[25]Ji C, Cao C, Lu S, Kivlin R, Amaral A, Kouttab N, Yang H, Chu W, Bi Z, Di W, Wan Y. Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells. Cancer Chemother Pharmacol.2008; 62(5):857-62.
[26]Jablonski EM, Mattocks MA, Sokolov E, Koniaris LG, Hughes FM Jr, Fausto N, Pierce RH, McKillop IH. Decreased aquaporin expression leads to increased resistance to apoptosis in hepatocellular carcinoma. Cancer Lett.2007; 18; 250(1):36-46.
[27]Jablonski EM, Webb AN, McConnell NA, Riley MC, Hughes FM Jr Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease.Am J Physiol Cell Physiol.2004; 286(4):C975-85.
[28]Monzani E, Shtil AA, La Porta CA.The water channels, new druggable targets to combat cancer cell survival, invasiveness and metastasis. Curr Drug Targets. 2007;8(10):1132-7. Review.
[29]Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature. 2005; 434(7034):786-792.
[30]Baluk P, Hashizume H, McDonald DM. Cellular abnormalities of blood vessels as targets in cancer. Curr. Opin. Genet. Dev.2005; 15:102-111.
[31]Mobasheri A, Airlev R, Hewitt SM, Marples D. Heterogeneous expression of the aquaporin 1 (AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung:a study using high density multiple human tumor tissue microarrays. Int. J. Oncol.2005; 26(5):1149-58..
[32]Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels. Nat Rev Cancer.2005; 5:423-435.
[33]Reese J, Das SK, Paria BC, Lim H, Song H,Matsumoto H, Knudtson KL, DuBois RN, Dey SK.Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. Biol. Chem.2001; 276(47): 44137-45.
[34]Rubanyi GM, Johns A, Kauser K. Effect of estrogen on endothelial function and angiogenesis. Vasc. Pharmacol.2002; 38(2):89-98.