【作者】 张丽；

【导师】 张雷；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2018， 硕士

【摘要】 目的:本研究通过对人正常子宫内膜细胞及子宫内膜异位症患者在位子宫内膜细胞进行体外培养,应用不同浓度IL-17A作用于细胞,模拟异位症微环境,观察其对人子宫内膜细胞增殖情况的影响。方法:1采用胶原酶I消化法,体外分离与培养人子宫内膜上皮细胞(endometrial epithelial cells,EECs)及子宫内膜基质细胞(endometrial stromal cells,ESCs),使用倒置相差显微镜观察人子宫内膜细胞的形态和生长状态;用免疫细胞化学的方法鉴定人子宫内膜上皮细胞和人子宫内膜基质细胞。2用免疫化学方法检测人子宫内膜细胞的IL-17A受体的表达情况。3用MTT法观察不同浓度IL-17A作用24h对人正常EECs、ESCs和EM患者在位EECs、ESCs增殖的影响,以挑选出最佳作用浓度进行后续实验。4采用实时荧光定量PCR方法检测0μg/ml(对照组)、250μg/ml(实验组)IL-17A作用于EM患者在位子宫内膜细胞后其PCNA基因mRNA表达水平,进行统计学比较。5应用Western-blot检测0μg/ml(对照组)、250μg/ml(实验组)IL-17A作用于EM患者在位子宫内膜细胞后β-catenin蛋白的表达水平,进行统计学比较。结果:1.光镜下EM患者在位子宫内膜与人正常子宫内膜细胞形态学无显著差异。EECs贴壁生长后,呈洋葱样或团块状排列。免疫细胞化学实验可见胞质部分角蛋白抗体(Cytokeratin)染色呈棕黄色,为阳性,细胞核不着色,为阴性,苏木精染色后呈紫红色;波形蛋白抗体(Vimentin)染色阴性。ESCs大部分以分散的单个细胞形式存在,贴壁生长后呈多角形,排列成束。免疫细胞化学可见胞质部分Vimentin染色呈棕黄色,细胞核不显色,苏木精染色后呈紫红色;Cytokeratin染色阴性。2.人正常EECs和ESCs和EM患者在位EECs和ESCs IL-17A受体免疫细胞化学检测结果均显示细胞胞质呈棕黄色,阳性表达;细胞核不着色,为阴性。3.经不同浓度IL-17A处理人正常子宫内膜细胞24h以后,MTT检测各实验组与对照组的差异均无统计学意义,P>0.05。4.经不同浓度IL-17A处理EM患者在位子宫内膜细胞24h以后,各实验组OD值与0μg/ml组OD值分别比较,除50μg/ml组与0μg/ml组差异无统计学意义外,其余各实验组OD值较对照组均升高,差值具有统计学意义(P<0.05),250μg/ml IL-17A处理组与对照组统计学差异最明显。实验组100μg/ml、250μg/ml、500μg/ml,700μg/ml之间比较,其差值无统计学意义。5.经终浓度0μg/ml(对照组)、250μg/ml(实验组)IL-17A分别处理EM患者在位子宫内膜细胞24h以后,RT-PCR结果为:EECs和ESCs的实验组的PCNA mRNA相对表达量均较对照组升高,P<0.05。6.Western-blot结果显示:实验组的EM患者在位子宫内膜上皮细胞和基质细胞的β-catenin蛋白相对表达量均较对照组升高,P<0.05。结论:1.子宫内膜异位症患者在位子宫内膜细胞与人正常子宫内膜细胞在形态学上并无肉眼可见的显著差异。2.IL-17A对人正常子宫内膜细胞的增殖无影响。3.IL-17A能促进子宫内膜异位症患者的在位子宫内膜上皮细胞和基质细胞PCNA mRNA的相对表达量升高。4.IL-17A能促进子宫内膜异位症患者的在位子宫内膜上皮细胞和基质细胞β-catenin的相对表达量升高。5.IL-17A能促进子宫内膜异位症患者在位子宫内膜细胞的增殖,可能参与了子宫内膜异位症的发生发展。更多还原

【Abstract】 Objective: In this study,the Interleukin-17 A in different concentrations was applied to the endometrial cells cultured in vitro from normal endometrial tissues and EM patients’ eutopic endometrial tissues to observe the effect of IL-17 A on proliferation of human endometrial cells in the simulative microenvironment of endometriosis.Methods:1 The human endometrial epithelial cells(EECs)and endometrial stromal cells(ESCs)were isolated and cultured in vitro by collagenase I digestion.The morphology and growth status of the cells was observed using the inverted phase contrast microscope.EECs and ESCs were identified by immunocytochemistry.2 The IL-17 A receptors on human endometrial cells were detected by immunochemistry.3 The IL-17 A in different concentrations was applied to EECs and ESCs for 24 h to observe the effect on proliferation of the cells.The MTT experiments were performed to select the optimal concentration for subsequent experiments.4 The mRNA of PCNA gene in EM patients’ eutopic endometrial cells treated by IL-17 A in the concentrations of 0μg/ml and 250μg/ml was detected by real-time PCR and compared by statistical analysis.5 The expression of β-catenin in EM patients’ eutopic endometrial cells treated by IL-17 A in the concentrations of 0μg/ml and 250μg/ml was detected by western-blot and compared by statistical analysis.Results:1.There was no significant difference in the morphology of normal endometrial cells and EM patients’ eutopic endometrial cells.The adherentEECs grew in the form of onion or crumb.The results of immunocytochemistry showed that the Cytokeratin in cytoplasm was stained to claybank,which was positive.The non-staining nucleus was stained purple by hematoxylin.And the stain of Vimentin was negative.Most of the ESCs dispersed to single cells.The adherent cells in the form of polygon were arranged in bundles.The results of immunocytochemistry showed that Vimentin in cytoplasm was stained to claybank.The non-staining nucleus was stained purple by hematoxylin.The stain of Cytokeratin was negative.2.The results of immunocytochemistry of EECs and ESCs from normal endometrial tissue and EM’s eutopic endometrial tissue showed that the cytoplasm was claybank,which indicated positive expression.The nucleus was nonstaining,which was negative.3.There was no significant difference between each treatment and control groups in MTT by treating human normal endometrial cells for 24 h using IL-17 A in different concentrations,P>0.05.4.The endometrial cells from EM patients were treated by IL-17 A in different concentrations for 24 h.The OD in each treatment groups was compared with that in control group.The OD in each treatment group was significantly higher than that in control group except for the group of IL-17 A in 50μg/ml(P<0.05).The OD differences between treatment groups were not significant.5.The results of RT-PCR showed that the level of PCNA mRNA in EECs and ESCs in the treatment group was significant higher than that in control group(P<0.05).6.The western-blot results showed that the expression of β-catenin in EECs and ESCs in treatment groups were significantly higher than that in control group(P<0.05).Conclusions:1.There is no visible difference in the morphology of normal endometrial cells and EM patients’ eutopic endometrial cells.2.IL-17 A has no effect on normal human endometrial cell proliferation.3.IL-17 A can promote the expression of PCNA mRNA in the epithelial cells and stromal cells from EM patients’ eutopic endometrial tissues.And the expression of PCNA mRNA in stromal cells is higher than that in epithelial cells.4.IL-17 A can promote the expression of β-catenin in the epithelial cells and stromal cells in EM patients’ eutopic endometrial tissues.And the expression of β-catenin in stromal cells is higher than that in epithelial cells.5.IL-17 A can promote the proliferation of EM patients’ eutopic endometrial cells and may be involved in the development of endometriosis..更多还原