【作者】 徐小敏；

【导师】 林俊；

【作者基本信息】 浙江大学 ， 妇产科学， 2017， 硕士

【摘要】 研究背景:子宫内膜异位症(简称内异症,Endometriosis,EMs)是具有生长功能的子宫内膜增殖(腺体和间质)出现在子宫腔被覆内膜及宫体以外的其他部位而引起的病症,是育龄妇女的常见病、多发病。据不同的研究人群,EMs在无症状女性中患病率为2～22%,在不孕患者中发病率高达20%～50%,盆腔痛患者中发病率40～50%。内异症是有着侵袭、转移、复发恶性生物学行为的良性疾患,在远处转移和种植生长能力诸多方面与恶性肿瘤有极大相似性。内异症的发病机制至今不清,目前以种植学说为主导理论。研究发现子宫内膜碎片(腺上皮及间质细胞)必须通过粘附、侵袭、血管生成才能生存、生长,引起病变和症状。多肽N-乙酰氨基半乳糖转移酶(UDP-N-acetyl-a-D-galactosamin:polypeptide N-acetylgalactosaminyltransferases,Ga；NAc-T)的异常表达,可以改变酶活性以及底物特异性。而异常糖基化则是许多常见肿瘤中的细胞生长、分化、黏附、转移以及肿瘤免疫监视的普遍现象。GalNAc-T3(polypepti-N-acetylgalactosaminyltransferase3,GalNAc-T3)及GalNAc-T6 是 GalNAc-Ts 家族中的成员,在多种肿瘤中均有表达,我们推测GalNAc-T3及GalNAc-T6的异常表达,可能改变了子宫内膜细胞的异位黏附和侵袭,从而促进内异症的发生和发展。研究目的:通过检测EMs患者的异位子宫内膜、在位子宫内膜以及非EMs患者的正常子宫内膜组织中Ga；NAc-T3和Ga；NAc-T6的表达情况,探讨GalNAc-T3和Ga；NAc-T6在子宫内膜异位症中的表达及在不同期别的相关性。材料与方法:收集内异症(根据r-AFs分期,其中Ⅰ-Ⅱ期6例,Ⅲ-Ⅳ期6例)手术患者,术后经病理证实为卵巢子宫内膜异位囊肿,取其异位子宫内膜及在位子宫内膜组织作为研究组,选择同期因纵隔子宫行宫腹腔镜联合检查并行宫腔镜纵膈切除术,术后经病理证实为正常子宫内膜的、排除盆腔子宫内膜异位症的患者,收集其正常内膜作为对照组。通过Real-time RT-PCR检测GalNAc-T3和GaINAc-T6 mRNA的表达;通过Western blot方法对GalNAc-T3和GalNAc-T6蛋白在不同子宫内膜中的表达进行半定量分析;通过免疫组化方法对在异位内膜、在位内膜、正常内膜中GalN Ac-T3和GalN Ac-T6蛋白的表达进行定位以及半定量分析。结果:1.Imunohistochemistr结果:在EMs患者的异位内膜、在位内膜以及正常子宫内膜组织中均有GlalN Ac-T3和GalN Ac-T6表达,阳性表达主要定位在子宫内膜腺细胞的细胞膜和细胞质。GalNAc-T3和GalNAc-T6表达的强弱顺序均为:异位子宫内膜组<对照子宫内膜组,组间差异有统计学意义(P<0.05)。2.Real-timeRT-PCR结果:GalNAc-T3和GalNAc-T6 mRNA表达为:正常子宫内膜组>在位子宫内膜组>异位宫内膜组,GalNAc-T3和GalNAc-T6在异位内膜的表达显著低于在位内膜及正常内膜,差异有统计学意义(P<0.05),但两者在在位内膜及正常内膜中表达差异无明显统计学意义(P>0.05)。3.Western blot结果:GalNAc-T3和GalNAc-T6表达的表达情况:异位内膜组的表达最低,在位内膜组次之,正常子宫内膜组表达最高,GalNAc-T3和GalNAc-T6在异位内膜的表达显著低于在位内膜及正常内膜,差异有统计学意义(P<0.05),但两者在在位内膜及正常内膜中表达无明显统计学差异(P>0.05)。4.GalNAc-T3和GalNAc-T6在各组内膜中均有表达,且不受月经周期影响。处于Ⅲ/Ⅳ期患者的异位内膜的GalN Ac-T3和GalN Ac-T6的表达明显低于Ⅰ/Ⅱ期患者。结论:1.GalNAc-T3及GalNAc-T6的低表达,可能增加了异位内膜细胞间的粘附和侵袭,从而促进了子宫内膜异位症的发生发展。2.GalNAc-T3及GalNAc-T6的低表达与内异症的分期存在密切的相关性。更多还原

【Abstract】 Background:Endometriosis(hereinafter referred to as the different disease,Endometriosis,EMs)is a common gynecological disorder characterized by the presence of endometrial glands and stroma outside of the uterine cavity.EMs is a common disease and frequently encountered disease in women of childbearing age.According to different research groups,EMS in asymptomatic women prevalence for,2～22%in patients with infertility rates as high as 20%～50%,the incidence of a disease in the patients with pelvic pain is 40～50%,is regarded as "the modern disease".Although EMs is a benign disease,it has great similarity with cancer in the invasion,angiogenesis,proliferation,apoptosis and many other aspects.However,the pathogenesis is controversial.The implantation hypothesis of Sampson is widely accepted.Previous study has confirmed that fragments of the endometrium survive,grow and cause symptoms by adhesion,invasion and angiogenesis.The study found that endometrial debris(gland epithelial and mesenchymal cells)must pass the adhesion,invasion and angiogenesis survival,growth,cause disease and symptoms.Polypeptide N-acetyl amino galactosyl transferase(UDP-N-acetyl-a-D-galactosamin:polypeptide N-acetylgalactosaminyltransferases,GalNAc)family is catalytic O-the first step in the process of sugar chain formed reaction.Has tumor associated antigen,one reason is that abnormal expression of glycosyl transferase,lead to the change of the enzyme activity and change of substrate specificity.Abnormal glycosylation in many common tumor cell growth,differentiation,adhesion,and tumor immune surveillance has played a very important role.GalNAc-T3 and GalNAc-T6 is the member in the family,are expressed in a wide variety of tumor,we hypothesized that GalNAc-T3 and the expression of GalNAc-T6,may change the ectopic endometrial cells adhesion,promoted the development of endometriosi.Objective:To investigate the expression patterns of N-acetyl galactosamine transferases(GalNAc-Ts)3 and GalNAc-T6 in clinicopathologically characterized endometriosis(EMs),and to explore their clinical significance.Materials and Methods:We obtained ovarian endometriomas and endometrial tissues from patients with confirmed EMS(n=12),whereas normal control endometrium was obtained from patients with uterine septum(n=12).The mRNA and protein levels of GalNAc-T3 and GalNAc-T6 were detected in these samples using quantitative real-time PCR,immunohistochemistry,and western blotting.Result:1.Immunohistochemistry results:GalNAc-T3 and GalNAc-T6 in EMs ectopic group,eutopic endometrium and normal tissue were expressed,mainly in the endometrial glands and glandular epithelium on the cell membrane and cytoplasm.The expression levers of GalNAc-T3 and GalNAc-T6:control endometrium>eutopic tissue>ectopic group,the expression was significantly lower with ectopic group compared to eutopic and control group(P<0.05);whereas the expression between eutopic and control tissue had no significant differences.(P>0.05).2.Real-time RT-PCR results:The expression of GalNAc-T3 and GalNAc-T6 mRNA:control endometrium>eutopic tissue>ectopic group,the expression was significantly lower with ectopic group compared to others(P<0.05),whereas the expression between eutopic and control group had no significant differences(P>0.05).3.Western blot results:The expression levers of GalNAc-T3 and GalNAc-T6:ectopic tissue<eutopic endometrium<control group,the expression was significantly lower with ectopic group compared to others(P<0.05);However,the expression of GalNAc-T3 and GalNAc-T6 between eutopic and control endometrium had no significant differences(P>0.05).4.GalNAc-T3 and-T6 were expressed in the endometrium of all groups,with no significant changes observed during the menstrual cycle.Furthermore,the expression of GalNAc-T3 and-T6 was significantly lower in patients with stage Ⅲ/Ⅳ EMS compared to patients with stage Ⅰ/Ⅱ(P<0.05).Conclusion:1.Both GalNAc-T3 and-T6 expression was downregulated in ectopic endometrium,which may increase the adhesion and invasion of endometrial cells and contribute to the development of EMS.2.Moreover,we found a strong correlation between the expression of GalNAc-T3 and-T6 and different stages of EMS.更多还原