【作者】 周俊杰；

【导师】 孔红霞；

【作者基本信息】 郑州大学 ， 妇产科学（专业学位）， 2017， 硕士

【摘要】 随着人们对生活质量要求的提高,困扰人们身心健康的不仅仅是肿瘤,子宫腺肌病所引起的不孕与痛经等症严重影响广大女性的生活质量。据文献报道,子宫切除术后的病理结果经数据统计显示,子宫腺肌病的发病率为8%-27%,在30-50岁妇女较为常见。虽然子宫腺肌病的诊治水平不断提高,但其发病率有持续上升、发病年龄日益年轻化趋势。子宫腺肌病的病因多且复杂,涉及多次妊娠、分娩、人工流产等,目前子宫腺肌病的发病机制尚未达成一致。随着影像学技术的不断发展,核磁共振(MRI)发现子宫结合带(JZ)呈低信号强度且厚度超过12mm,可用于临床诊断子宫腺肌病,其准确率达85%。有研究认为子宫腺肌病的发生与子宫结合带结构的破坏密切相关。因此寻找子宫结合带上与子宫腺肌病发展有关的调节因子,通过生物学作用恢复子宫结合带正常结构从而抑制子宫腺肌病发生发展,将成为国内外学者研究的热点问题。G蛋白偶联受体30(GPR30)是作为7次跨膜G蛋白偶联受体超家族的成员之一。可与雌激素结合产生快速非基因组效应,传递化学信息或神经冲动,激发下游效应因子,活化多种信号转导途径,在相应的靶组织中导致细胞增殖。雌激素与GPR30结合后,可引发EGFR/MAPK/ERK等级联反应,促使细胞的增殖、分化、迁移等;可通过PI3K/Akt途径促进细胞增殖;也可通过调节胞内第二信使如cAMP、Ca2+等的变化,将胞外信号传递到胞内,实现细胞的生物学作用。GPR30不仅参与调节人体正常生理功能,而且与多种雌激素依赖性疾病的发生有着紧密联系。表皮生长因子受体(EGFR)是酪氨酸激酶受体家族的成员之一,可与多种配体结合,活化胞内酪氨酸激酶区,启动多条信号转导通路,将胞外信号转化成胞内信号,参与细胞的增殖、分化和凋亡等多种重要的生物学过程。GPR30介导PI3K/Akt通路诱发子宫内膜癌细胞的增殖需要EGFR的反式激活。EGFR作为多条信号通路的枢纽,在多种雌激素依赖性肿瘤的发病机制中发挥着关键作用。本研究试图通过检测GPR30和EGFR在子宫腺肌病子宫结合带组织中的表达变化,并探讨其与子宫腺肌病发病的关系,为GPR30/EGFR信号途径在子宫腺肌病中的作用提供研究基础。目的本实验采用免疫组化法和实时荧光定量PCR方法检测处于子宫内膜增生期及分泌期的子宫腺肌病患者子宫结合带(JZ)组织中GPR30和EGFR的表达情况,分析二者在子宫腺肌病子宫结合带与正常子宫结合带组织中的表达差异,探讨其在子宫腺肌病发生发展中的作用,为研究GPR30信号转导通路与子宫腺肌病子宫结合带的关系提供依据。方法84例行子宫全切术或子宫次全切术的患者中,50例子宫腺肌病患者(子宫腺肌病组,28例留取处于子宫内膜增生期的JZ组织、22例留取处于子宫内膜分泌期的JZ组织),34例宫颈病变患者(对照组,18例留取处于子宫内膜增生期的子JZ组织、16例留取处于子宫内膜分泌期的JZ组织)。JZ组织根据MRI检查定位并获取,采用免疫组化二步法检测GPR30蛋白、EGFR蛋白在子宫腺肌病组和对照组的JZ组织中的表达情况,采用实时荧光定量PCR法检测GPR30 mRNA、EGFR mRNA在两组JZ组织中的相对表达量。统计学方法采用SPSS22.0软件进行分析。定量资料采用x±s表示,组间比较采用t检验或校正t检验;定性资料采用χ2检验或校正χ2检验。GPR30和EGFR在子宫腺肌病子宫结合带组织中的关联性分析采用二分类χ2检验。检验水准α=0.05。结果1.GPR30、EGFR在细胞膜和细胞质中均有表达。GPR30、EGFR在子宫腺肌病组JZ组织中表达明显高于对照组,差异均有统计学意义(P<0.05)。2.GPR30、EGFR在对照组JZ组织中有周期性变化,即子宫内膜增生期的阳性表达率高于分泌期,差异均有统计学意义(P<0.05);GPR30、EGFR在子宫腺肌病组JZ组织中无周期性变化,子宫内膜增生期阳性表达率与分泌期相比,差异无统计学意义(P>0.05)。3.GPR30 mRNA、EGFR mRNA在子宫腺肌病JZ组织中相对表达量高于对照组,差异有统计学意义(P<0.05)。4.GPR30 mRNA、EGFR mRNA在对照组JZ组织中有周期性变化,即子宫内膜增生期表达高于分泌期(P<0.05)。GPR30 mRNA、EGFR mRNA在子宫腺肌病组JZ组织中无周期性变化,子宫内膜增生期与分泌期相比,差异无统计学意义(P>0.05)。5.GPR30与EGFR在子宫腺肌病JZ组织中的表达呈正相关。结论1.GPR30和EGFR在正常子宫结合带组织中有周期性改变,该变化可能受卵巢激素调控。2.GPR30和EGFR在子宫腺肌病子宫结合带组织中高表达且无周期性改变,该变化可能与子宫腺肌病的发病有关。3.GPR30和EGFR相互作用共同参与子宫腺肌病的发生发展。更多还原

【Abstract】 With the improvement of people’s quality of life,it is not only the tumor suffering people’s physical and mental health but also infertility and dysmenorrheathe by the adenomyosis,which affect the quality of women’ life.According to reports in the literature,the pathological results after hysterectomy by data statistics show that the incidence of adenomyosis ranging from 8% to 27%,which is more common in women aged between 30 to 50 years old.Although the level of diagnosis and treatment of adenomyosis has been increasing,its morbidity has been increasing and the age of onset has been younger and younger.The etiology of adenomyosis is complex,involving pregnancy,production,and it may be related with hormonal disorders,endometrial basal invagination,lymphatic and venous spread,immune factors and genetic changes.While the mechanism has not been yet reached a consensus.With the development of imaging technology,magnetic resonance imaging has seen a signal of low intensity and a thickness of more than 12 millimeters,which can be used for clinical diagnosis of adenomyosis,and the accuracy rate is 85%.It is suggested that adenomyosis is caused by the damage of the structure and function of the uterine junctional zone.Therefore,it is a hot issue in the world to find out the regulatory factor in the uterine junctional zone which is connected with the development of adenomyosis and we may try to restore the structure of the uterine junctional zone to inhibit the adenomyosis by biological effects.G protein-coupled receptor 30(GPR30),as a member of the 7 transmembrane G protein coupled receptor superfamily,GPR30 is involved in the rapid nongenomic effect,by transmiting chemical cues or nerve impulse,thus affecting the downstream effector molecules to active many signal transduction pathways and exert their biological effects,such as cell cycle progression or cell proliferation in the corresponding target tissue.When estrogen integrates with GPR30,cascade reaction is induced by the EGFR/MAPK/ERK,to promote cell proliferation and differentiation;or by the pathway of PI3K/Akt to promote cell proliferation;or by regulating the intracellular second messengers such as cAMP and Ca2+,to transfer extracellular signal to intracellular cells.GPR30 is not only involved in regulating the normal physiological function of human body,but also closely related to the occurrence of the hormone-dependent diseases.Epidermal growth factor receptor(EGFR),as one of the receptor tyrosine kinase family members,can be combined with a variety of ligands,resulting in intracellular tyrosine kinase activation,thereby activating multiple signal transduction,transfering extracellular signals into intracellular signals and affecting cell proliferation,differentiation and apoptosisin many important biological processes.GPR30 mediates the PI3K/Akt pathway in endometrial carcinoma cells under the trans activation of EGFR.As the intersection of many different signaling pathways,EGFR plays an important role in the pathogenosis and etiotogy of many estrogen-dependent tumors.This study attempts to explore the effect of GPR30 and EGFR in the uterine junctional zone of adenomyosis by detecting their expression in the tissue,and provide the research foundation for GPR30/EGFR signal pathway in adenomyosis.ObjectiveThis experiment detects the expression of GPR30 and EGFR in JZ of patients with adenomyosis in the endometrial proliferative and secretory phase by immunohistochemistry and real-time fluorescence quantitative PCR method.And we analyze the differences between adenomyosis and normal JZ tissues and research the effect in the occurrence and progress of adenomyosis,in order to providing the basis for the study on the relationship between the GPR30 signal transduction pathway and JZ tissues of adenomyosis.Methods84 cases underwent hysterectomy or subtotal hysterectomy patients,are involved in 50 cases of adenomyosis patients(adenomyosis group,28 cases of specimens from JZ in the endometrial proliferative phase and 22 cases from JZ in the endometrial secretory phase),and 34 cases of patients with cervical lesions(control group,18 cases of specimens from JZ in the endometrial proliferative phase and 16 cases from JZ in the endometrial secretory phase).JZ tissues are located and obtained in both groups by MRI.GPR30 and EGFR protein are detected by immunohistochemical method in JZ of adenomyosis group and the control one.The expressions of GPR30 and EGFR mRNA in JZ of both groups are detected by real-time fluorescent quantitative PCR.Statistical methodsUsing SPSS22.0 statistical software for analysis.Quantitative datas are presented in the form of x ±s,and different groups are compared by t test or corrected t test;Qualitative datas are analyzed by Chi-Square Tests or corrected Chi-Square Tests.Chi-Square Tests are used to analyze the relationship between GPR30 and EGFR in uterine junctional zone of adenomyosis.Inspection standard α= 0.05.Results1.GPR30 and EGFR are both expressed in the cell membrane and cytoplasm.The expressions of GPR30 and EGFR in the JZ of adenomyosis group are observably higher than that in the control group,which the differences are statistically significant(P<0.05).2.In the control group,the positive rates of GPR30 and EGFR in JZ have periodic variational regularities,that in the endometrial proliferative phase are higher than the endometrial secretory phase,and the differences are statistically significant(P<0.05);however,in JZ of the adenomyosis group the positive rates of GPR30 and EGFR are non-periodic variational,which is no significant difference in endometrial proliferative phase compared with that in endometrial secretory phase(P>0.05).3.The relative expressions of GPR30 mRNA and EGFR mRNA in JZ of adenomyosis are higher than that in the control group,which the differences are both statistically significant(P<0.05).4.In the control group,the expressions of GPR30 mRNA and EGFR mRNA in JZ have periodic variational regularities,that in the endometrial proliferative phase are higher than the endometrial secretory phase,which the differences are statistically significant(P<0.05);in the adenomyosis group,the expressions of GPR30 mRNA and EGFR mRNA in JZ are non-periodic variational,which is no significant difference in endometrial proliferative phase compared with that in endometrial secretory phase(P>0.05).5.The expressions of GPR30 and EGFR in JZ of adenomyosis are positively correlated.Conclusions1.In the normal uterine junctional zone,the expressions of GPR30 and EGFR both have periodic variational regularities,which the changes may be regulated by ovarian hormone.2.In the uterine junctional zone of adenomyosis,the high expressions of GPR30 and EGFR are non-periodic variational,which may be related to the pathogenesis of adenomyosis.3.In adenomyosis,GPR30 and EGFR may be related to the occurrence and progress together.更多还原