ADAR1基因在子宫内膜异位性疾病的在位及异位内膜组织中的表达及意义

发布时间：2018-02-02 08:09

  本文关键词： ADAR1基因 子宫内膜异位症(EMs) 子宫腺肌症(AM)　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：子宫内膜异位症(endometriosis,EMs)简称内异症,是一种很常见的妇科疾病,以子宫内膜样组织生长在子宫以外为特征,如卵巢,输卵管,盆腔和腹腔,其中以异位于卵巢最常见。子宫腺肌病(adenomyosis,AM)是指具有生长功能的子宫内膜腺体和间质侵入子宫肌层,以月经量过多,经期延长及继发性痛经为主要临床表现的常见妇科疾病。AM被认为是子宫内膜异位症的一种,其发病机制存在一定的相似性。EMs和AM被描述为非肿瘤性的疾病,但其在生物学行为上却呈现出与恶性肿瘤一致的特性,如可异常增殖、侵润甚至侵袭。近年的研究还表明,细胞免疫和体液免疫功能失调均可能与Ems和AM的发生发展有关。国内外有关ADAR1与子宫内膜异位症和子宫腺肌病发病关系的研究未见报道。ADAR1是一种RNA编辑酶,RNA编辑是指DNA转录成RNA后对RNA的修饰和加工,可能使基因在DNA水平的表达与其在蛋白水平的表达完全不同,从而改变了遗传信息[2-6]。研究发现ADAR1在细胞增殖[7]、分化[8]及肿瘤发展[9]中起了十分重要的作用。此外,ADAR1与炎症和免疫也关系密切[10]。本实验采用实时荧光定量PCR,Western blotting及免疫组织化学等方法检测ADAR1在内异症和腺肌病患者在位内膜与异位内膜及腺肌瘤组织中的表达,进一步探讨两种疾病发病机制以期对临床诊治提供新的思路。目的研究ADAR1在子宫内膜异位症和子宫腺肌病中的表达差异,探讨ADAR1与子宫内膜异位症和子宫腺肌病发病的关系及意义。资料与方法该研究经郑州大学第三附属医院即河南省妇幼保健院伦理委员会批准,所有患者均签署知情同意书。1研究对象选取2015年7月至2016年10月在河南省郑州大学第三附属医院(即河南省妇幼保健院)妇科行手术治疗的EMs患者31例,年龄29~44岁,手术证实为子宫内膜异位症III型或IV型(按美国生殖医学协会定义),对其中有生育要求的25例患者同时行宫腔镜检查,诊刮得到子宫内膜组织(实验组EU组),术中取异位内膜组织(实验组EC组)。选同期在河南省郑州大学第三附属医院就诊行子宫全切的子宫腺肌病患者30例,年龄38~48,子宫全切后立即取患者在位子宫内膜组织(实验组AM1),及腺肌瘤组织(实验组AM2);对照组为同期子宫肌瘤行子宫全切的患者30例,年龄34~49岁,子宫全切后立即取在位子宫内膜组织(对照组C)。上述患者月经规律,26~32天;术前六个月无激素及内分泌疾病用药史,无妊娠、哺乳,术前无绝经期症状。以上标本均在子宫内膜增殖期获取,且全部经病理证实。2标本处理在无菌状态下获取的子宫内膜组织或肌瘤组织,一半立即放入液氮,后转入-80℃保存用于实时荧光定量PCR和蛋白印记实验,另一半放入10%中性福尔马林溶液固定后石蜡包埋,用于组织学检查。3试验方法2.1实时荧光定量PCR技术(qRT-PCR)检测各组中ADAR1mRNA的表达。2.2 Western blotting半定量检测各组ADAR1蛋白表达水平。2.3免疫组化对各组ADAR1蛋白表达进行定位及半定量分析。4统计学分析实验数据以均数±标准差(x±s)表示,采用SPSS 21.0软件进行统计分析,组间两两比较用t检验,满足正态性和方差齐性的多组比较采用单因素方差分析,若不满足则采用K个独立样本检验或Kruskal-Wallis秩和检验。检验水准α=0.05。两两比较采用Bonferroni法,以Pα为差异有统计学意义。结果1各组年龄比较子宫内膜异位症组(36.4±3.8),子宫腺肌病组(39.13±5.61)和对照组(40.3±3.4),组间无统计学差异(P0.05)。2 ADAR1mRNA在子宫内膜异位症和子宫腺肌病中的表达实时荧光定量PCR结果显示ADAR1 P150mRNA在EC(2.33±0.28)组和EU(2.18±0.36)组的表达明显高于C(1.68±0.43)组(P0.05),但EU组和EC组ADAR1 P150 mRNA的表达差别无统计学意义,且ADAR1 P110 mRNA在子宫内膜异位症EU组(1.01±0.21),EC组(1.01±0.15)和C组(0.99±0.11)的表达差别无统计学意义(P0.05)。与EMs组结果相似,实时荧光定量PCR结果显示ADAR1 P150 mRNA的表达在AM1组(2.09±0.26)和AM2组(2.09±0.25),均高于对照组(1.68±0.43)差异有统计学意义(P0.05);而ADAR1 P110 mRNA的表达在AM1组(0.97±0.21),AM2组(0.87±0.17),C组(0.99±0.11)差异无统计学意义(P0.05)。3 ADAR1蛋白在子宫内膜异位症和子宫腺肌病中的表达Western Blotting的结果显示ADAR1蛋白的表达在EU组(0.39±0.05)和EC(0.40±0.07)组的均明显高于C组(0.104±0.04)(P0.05),且EU和EC两组ADAR1蛋白的表达差异无统计学意义(P0.05)。ADAR1蛋白表达在AM1组(0.34±0.06)和AM2组(0.35±0.06)均高于C组(0.104±0.04),差异均有统计学意义(P0.05)。AM1组的ADAR1蛋白表达与AM2组相比,差异无统计学意义(P0.05)。4 ADAR1蛋白在子宫内膜异位症和子宫腺肌病中的定位与表达免疫组化的结果显示ADAR1蛋白在内异症在位、异位内膜,腺肌瘤及在位内膜,对照组内膜的细胞胞浆中均可见棕黄色阳性染色。子宫内膜异位症组中ADAR1在位内膜(EU组),异位内膜(EC组)和正常内膜(C组)表达水平分别为(87.29±11.87),(96.65±13.31),(26.08±8.49),差异有统计学意义(p0.05),子宫腺肌病组ADAR1于在位内膜(AM1),腺肌瘤(AM2)和正常内膜C组的表达水平分别为(68.77±11.20),(67.48±11.44),(26.08±8.49),差异有统计学意义(p0.05)。结论ADAR1基因可能与子宫内膜异位症和子宫腺肌病的发病及发生发展有一定关系。为诊治子宫内膜异位症和子宫腺肌病提供了新思路。
[Abstract]:Endometriosis (endometriosis, EMs) referred to as endometriosis is a common gynecological disease, with endometrial tissue outside the uterus growth characteristics, such as ovarian, tubal, pelvic and abdominal cavity, which is the most common ovarian adenomyosis (adenomyosis, AM) refers to the uterus the growth of endometrial glands and interstitial myometrium, with menorrhagia, menostaxis and.AM of common gynecological diseases of secondary dysmenorrhea is the main clinical manifestation is believed to be an endometriosis, its pathogenesis there are some similarities between.EMs and AM is described as non neoplastic the disease, but its biological behavior is consistent with the characteristic of malignant tumors, such as proliferation, invasion and invasion. In recent years, the study also showed that cellular immunity and humoral immunity disorders are likely to occurrence and development of Ems and AM are . the domestic and foreign related ADAR1 in endometriosis and adenomyosis pathogenesis has not been reported that.ADAR1 is a RNA editing enzyme, RNA editing is the modification and processing of the RNA transcription of DNA into RNA, may make the gene expression in the level of DNA expression and protein level in a completely different, so as to change the study found that the genetic information of [2-6]. ADAR1 in [7] cells, play a very important role in the differentiation of [8] and [9] in tumor development. In addition, ADAR1 with inflammation and immunity is also closely related to the experiment of [10]. by real-time quantitative PCR, Western blotting and immunohistochemical method to detect expression of ADAR1 in endometrium and endometriosis and adenomyosis in the reign of patients with endometriosis and adenomyosis, to further explore the pathogenesis of the two diseases in order to provide new ideas for the clinical diagnosis and treatment. Objective to study the ADAR1 in endometriosis and sub The difference in the expression of uterine adenomyosis, to explore the relationship between ADAR1 and endometriosis and adenomyosis and its significance. Materials and methods the study by the Third Affiliated Hospital of Zhengzhou University, Henan Provincial Maternal and child health hospital ethics committee approval, all patients signed the informed consent of.1 from July 2015 to October 2016 in the Affiliated Hospital of Henan Province third Zhengzhou University (Henan Provincial Maternal and child health hospital) 31 EMs patients in the treatment of gynecological surgery patients, aged 29~44 years, surgery confirmed endometriosis III type or IV type (as defined by the American Society for Reproductive Medicine), there are also 25 cases of hysteroscopy for patients with fertility requirements of the endometrial curettage obtained the organization (group EU), and the ectopic endometrial tissue during the operation (group EC). Selected uterine gland in the Affiliated Hospital of Zhengzhou University of Henan province from third hysterectomy muscle In 30 cases, patients aged 38~48, immediately after hysterectomy patients in eutopic endometrium (experimental group, AM1) and adenomyoma tissues (experimental group AM2); 30 cases in the control group for the same period of uterine fibroids hysterectomy patients, aged 34~49 years old, hysterectomy immediately after taking eutopic endometrium (control group C). The patients with regular menstrual cycle, 26~32 days; six months before the operation without hormone and endocrine disease medication history, pregnancy, lactation, pre menopausal symptoms. All the specimens were obtained in the proliferative phase of endometrium, and all pathologically confirmed.2 specimens processed in aseptic conditions of endometrium tissue or leiomyoma, half immediately placed in liquid nitrogen, and then transferred to -80 DEG C for preservation of real-time fluorescence quantitative PCR and Western blot experiments, the other half in 10% formalin fixed paraffin embedded, 2.1 for real-time.3 test method to examine the histology Quantitative PCR Technology (qRT-PCR) to detect the ADAR1 protein expression detected by semi quantitative.2.2 Western blotting groups ADAR1mRNA.2.3 expression level in each group by immunohistochemistry ADAR1 protein expression and localization analysis of.4 semi quantitative statistical analysis the experimental data to mean + standard deviation (x + s), were analyzed by SPSS 21 software. Between the 22 groups were compared with t test, meet the normality and homogeneity of variance were analyzed by single factor analysis of variance, if not satisfied with independent samples K test or Kruskal-Wallis rank sum test. The test level of alpha =0.05. 22 compared with Bonferroni method, using P alpha values. There was significant difference between the 1 the age group of endometriosis group (36.4 + 3.8), adenomyosis group (39.13 + 5.61) and control group (40.3 + 3.4), there was no significant difference between groups (P0.05).2 ADAR1mRNA in endometriosis and uterus 鑵鸿倢鐥呬腑鐨勮〃杈惧疄鏃惰崸鍏夊畾閲廝CR缁撴灉鏄剧ずADAR1 P150mRNA鍦‥C(2.33卤0.28)缁勫拰EU(2.18卤0.36)缁勭殑琛ㄨ揪鏄庢樉楂樹簬C(1.68卤0.43)缁，

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