Artemin基因在子宫内膜癌细胞增殖及侵袭中的作用
发布时间：2018-08-09 09:05
【摘要】：子宫内膜癌是女性生殖系统最常见的三大恶性肿瘤之一,近些年其发病率有上升趋势。大多数子宫内膜癌患者发病处于早期,经外科手术治疗预后较好;但仍有少数子宫内膜癌患者就诊时已处于疾病晚期,有报道晚期或经化疗复发患者的中位生存时间不超过1年。子宫内膜癌的发病类型可分为雌激素依赖型(Ⅰ型)和非雌激素依赖型(Ⅱ型),前者占内膜癌大多数,预后相对较好;后者因其生物特性具有侵袭性,具有早期转移的倾向,预后通常较差。Artemin(ARTN)是胶质细胞源性神经营养因子(gilal cell line derived neurotrophic factor,GDNF)家族中的一个成员,ARTN基因在体内广泛分布,提示它对多系统组织、器官的发育及生理功能的维持具有重要意义。ARTN基因同胰腺癌、食管癌、胃癌、乳腺癌等的发生、发展密切相关。本课题通过干扰及过表达ARTN基因,进一步研究ARTN基因对人子宫内膜癌细胞增殖、迁移及侵袭的影响。目的:从细胞水平探讨干扰及过表达ARTN基因对人子宫内膜癌细胞株的增殖、迁移及侵袭的影响,通过靶向干扰ARTN基因表达,为子宫内膜癌的靶向治疗提供一定理论依据。方法:1体外培养子宫内膜癌Ishikawa和HEC-1-A细胞,应用Western Blot方法检测两种细胞中ARTN蛋白表达情况。2以pGPU6/GFP/Neo为载体构建ARTN基因的短发夹RNA(pGPU6/GFP/Neo-ARTN shRNA)干扰质粒,以pcDNA3.1为克隆载体,构建ARTN基因的过表达质粒(pcDNA3.1-ARTN)。3将ARTN基因的干扰及过表达质粒用脂质体法转染人子宫内膜癌Ishikawa和HEC-1-A细胞,利用qRT-PCR及Western Blot法检测转染后两种细胞中ARTN mRNA及蛋白表达变化。4应用CCK法绘制细胞生长曲线,检测细胞增殖能力,同时利用transwell小室实验检测细胞迁移及侵袭能力变化。结果:1两种子宫内膜癌细胞中均有artn蛋白不同程度的表达,相对表达量分别为0.808±0.015和0.895±0.008。2转染pgpu6/gfp/neo-artnshrna干扰质粒后,两种细胞的artnmrna相对表达量明显下降(f=47.882,f=394.326,p0.05);转染pcdna3.1-artn过表达质粒后,两种细胞artnmrna相对表达量则明显增加(f=276.007,f=861.159,p0.05),差异有统计学意义。干扰artn基因表达后,两种细胞中的artn蛋白相对表达量均明显减少(f=726.323,f=1780.714,p0.05);强制表达artn基因后,两种细胞中的artn蛋白相对表达量则明显增加(f=1145.127,f=519.357,p0.05),差异均有统计学意义。3转染artn干扰质粒24h,两种细胞增殖能力无明显变化(p0.05),转染48h、72h、96h两种细胞增殖能力均明显降低(p0.05);转染artn过表达质粒24h,两种细胞增殖能力无明显变化(p0.05),转染48h、72h、96h两种细胞增殖能力均明显增强(p0.05)。4侵袭实验结果显示两种细胞干扰组比较阴性对照组和对照组穿膜细胞数明显减少,差异有统计学意义(f=55.609,f=108.865,p0.05);过表达组比较阴性对照组和对照组穿膜细胞数则明显增加,差异有统计学意义(f=42.880,f=32.004,p0.05)。迁移实验中两种细胞干扰组与阴性对照组、对照组比较,穿膜细胞数明显减少(f=52.938,f=90.224,p0.05);过表达组与阴性对照组、对照组比较,穿膜细胞数明显增加(f=196.899,f=100.167,p0.05),差异均有统计学意义;侵袭、迁移实验中将两种细胞的干扰组、过表达组和对照组穿膜细胞数进行对比,结果发现hec-1-a细胞比较ishikawa细胞各组穿膜细胞数都明显增加,差异均有统计学意义(p0.05)。结论:1子宫内膜癌ishikawa和hec-1-a细胞中均有不同强度的artn蛋白的表达。2小分子rna干扰技术可以有效抑制artn基因在mrna及蛋白质水平的表达;过表达技术则可增强artn基因的表达。3干扰ARTN基因表达可显著抑制子宫内膜癌细胞的增殖活性,抑制其迁移及侵袭能力;过表达则可显著增强这些作用。HEC-1-A细胞比Ishikawa细胞具有更强的侵袭及迁移能力。
[Abstract]:Endometrial carcinoma is one of the most common three malignant tumors in the female reproductive system. In recent years, the incidence of endometrial cancer is rising. Most patients with endometrial cancer are in the early stage, and the prognosis is better by surgical treatment. However, there are still a few endometrium cancer patients who are in the late stage of the disease, and there are reports of late or chemotherapy recurrent patients. The median survival time is not more than 1 years. The type of endometrial carcinoma can be divided into estrogen dependent (type I) and non estrogen dependent type (type II). The former accounts for most of the endometrial carcinoma, and the prognosis is relatively good. The latter has a tendency to metastasize early because of its biological characteristics, and the prognosis is usually poor.Artemin (ARTN) is the source of glial cell. A member of the gilal cell line derived neurotrophic factor (GDNF) family, ARTN gene is widely distributed in the body, suggesting that it is important for the maintenance of multi system tissue, organ development and physiological function. The.ARTN gene is closely related to the development of pancreatic cancer, food tube cancer, gastric cancer, breast cancer and so on. To further study the effect of ARTN gene on the proliferation, migration and invasion of human endometrial carcinoma cells by interfering and overexpressing ARTN gene. Objective: To investigate the effects of interference and overexpression of ARTN gene on the proliferation, migration and invasion of human endometrial cancer cells from the cell level, and to target endometrial carcinoma by targeting ARTN gene expression. The target therapy provided a certain theoretical basis. Methods: 1 Ishikawa and HEC-1-A cells in endometrial carcinoma were cultured in vitro, and the expression of ARTN protein in two cells was detected by Western Blot method. The RNA (pGPU6/GFP/Neo-ARTN shRNA) interference plasmid of ARTN gene was constructed by pGPU6/GFP/Neo as the carrier, and the pcDNA3.1 was used as the clone carrier. The overexpressed plasmid (pcDNA3.1-ARTN).3 of the TN gene transfected the ARTN gene and the overexpressed plasmid transfected into the human endometrial carcinoma Ishikawa and HEC-1-A cells by liposome method. The qRT-PCR and Western Blot methods were used to detect the ARTN mRNA and the protein expression changes in the two cells after the transfection. At the same time, the changes of cell migration and invasion were detected by Transwell chamber test. Results: 1 two kinds of endometrial carcinoma cells were expressed in different degrees of ARTN protein, and the relative expression of the relative expression was 0.808 + 0.015 and 0.895 + 0.008.2 transfected with pgpu6/gfp/neo-artnshrna interference plasmids respectively. The relative expression of artnmrna in the two cells was obviously lower. (f=47.882, f=394.326, P0.05); after transfection of pcdna3.1-artn overexpression plasmid, the relative expression of artnmrna in the two cells increased significantly (f=276.007, f=861.159, P0.05), and the difference was statistically significant. After the interference of the ARTN gene expression, the relative expression of ARTN protein in the two cells decreased significantly (f=726.323, f=1780.714,). After the N gene, the relative expression of ARTN protein in the two cells increased significantly (f=1145.127, f=519.357, P0.05), and the difference was statistically significant,.3 transfected ARTN interference plasmid 24h, the proliferation ability of the two cells was not significantly changed (P0.05), 48h, 72h, and 96h two cells were transfected, two kinds of plasmids were transfected. The proliferation ability of cell proliferation was not significantly changed (P0.05). The proliferation ability of two cells transfected with 48h, 72h, 96h was significantly enhanced (P0.05).4 invasion test results showed that the number of membrane cells in the two cell interference groups decreased significantly (f=55.609, f=108.865, P0.05) compared with the control group (f=55.609, f=108.865, and P0.05); the overexpressed group was compared with the negative control group and the control group. The number of membrane cells in the control group increased significantly (f=42.880, f=32.004, P0.05). In the migration experiment, the number of membrane cells decreased significantly (f=52.938, f=90.224, P0.05), and the number of membrane cells increased significantly (f=196.899, f=52.938, f=90.224, P0.05) compared with the control group. F=100.167, P0.05), the difference was statistically significant, the invasion, the migration experiment, the two cells in the interference group, the overexpressed group and the control group comparison of the number of membrane cells, the results showed that the number of HEC-1-A cells in each group of Ishikawa cell membrane cells increased significantly, the difference was statistically significant (P0.05). Conclusion: 1 endometrial carcinoma Ishikawa and H The expression of.2 small molecule RNA interference technique can effectively inhibit the expression of ARTN gene at the level of mRNA and protein in ec-1-a cells. The overexpression technology can enhance the expression of ARTN gene and interfere with the expression of ARTN gene, which can inhibit the proliferation activity of endometrial cancer cells and inhibit the migration and invasion ability of the endometrial cancer cells. Overexpression can significantly enhance these effects..HEC-1-A cells have stronger invasion and migration ability than Ishikawa cells.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
本文编号：2173600
[Abstract]:Endometrial carcinoma is one of the most common three malignant tumors in the female reproductive system. In recent years, the incidence of endometrial cancer is rising. Most patients with endometrial cancer are in the early stage, and the prognosis is better by surgical treatment. However, there are still a few endometrium cancer patients who are in the late stage of the disease, and there are reports of late or chemotherapy recurrent patients. The median survival time is not more than 1 years. The type of endometrial carcinoma can be divided into estrogen dependent (type I) and non estrogen dependent type (type II). The former accounts for most of the endometrial carcinoma, and the prognosis is relatively good. The latter has a tendency to metastasize early because of its biological characteristics, and the prognosis is usually poor.Artemin (ARTN) is the source of glial cell. A member of the gilal cell line derived neurotrophic factor (GDNF) family, ARTN gene is widely distributed in the body, suggesting that it is important for the maintenance of multi system tissue, organ development and physiological function. The.ARTN gene is closely related to the development of pancreatic cancer, food tube cancer, gastric cancer, breast cancer and so on. To further study the effect of ARTN gene on the proliferation, migration and invasion of human endometrial carcinoma cells by interfering and overexpressing ARTN gene. Objective: To investigate the effects of interference and overexpression of ARTN gene on the proliferation, migration and invasion of human endometrial cancer cells from the cell level, and to target endometrial carcinoma by targeting ARTN gene expression. The target therapy provided a certain theoretical basis. Methods: 1 Ishikawa and HEC-1-A cells in endometrial carcinoma were cultured in vitro, and the expression of ARTN protein in two cells was detected by Western Blot method. The RNA (pGPU6/GFP/Neo-ARTN shRNA) interference plasmid of ARTN gene was constructed by pGPU6/GFP/Neo as the carrier, and the pcDNA3.1 was used as the clone carrier. The overexpressed plasmid (pcDNA3.1-ARTN).3 of the TN gene transfected the ARTN gene and the overexpressed plasmid transfected into the human endometrial carcinoma Ishikawa and HEC-1-A cells by liposome method. The qRT-PCR and Western Blot methods were used to detect the ARTN mRNA and the protein expression changes in the two cells after the transfection. At the same time, the changes of cell migration and invasion were detected by Transwell chamber test. Results: 1 two kinds of endometrial carcinoma cells were expressed in different degrees of ARTN protein, and the relative expression of the relative expression was 0.808 + 0.015 and 0.895 + 0.008.2 transfected with pgpu6/gfp/neo-artnshrna interference plasmids respectively. The relative expression of artnmrna in the two cells was obviously lower. (f=47.882, f=394.326, P0.05); after transfection of pcdna3.1-artn overexpression plasmid, the relative expression of artnmrna in the two cells increased significantly (f=276.007, f=861.159, P0.05), and the difference was statistically significant. After the interference of the ARTN gene expression, the relative expression of ARTN protein in the two cells decreased significantly (f=726.323, f=1780.714,). After the N gene, the relative expression of ARTN protein in the two cells increased significantly (f=1145.127, f=519.357, P0.05), and the difference was statistically significant,.3 transfected ARTN interference plasmid 24h, the proliferation ability of the two cells was not significantly changed (P0.05), 48h, 72h, and 96h two cells were transfected, two kinds of plasmids were transfected. The proliferation ability of cell proliferation was not significantly changed (P0.05). The proliferation ability of two cells transfected with 48h, 72h, 96h was significantly enhanced (P0.05).4 invasion test results showed that the number of membrane cells in the two cell interference groups decreased significantly (f=55.609, f=108.865, P0.05) compared with the control group (f=55.609, f=108.865, and P0.05); the overexpressed group was compared with the negative control group and the control group. The number of membrane cells in the control group increased significantly (f=42.880, f=32.004, P0.05). In the migration experiment, the number of membrane cells decreased significantly (f=52.938, f=90.224, P0.05), and the number of membrane cells increased significantly (f=196.899, f=52.938, f=90.224, P0.05) compared with the control group. F=100.167, P0.05), the difference was statistically significant, the invasion, the migration experiment, the two cells in the interference group, the overexpressed group and the control group comparison of the number of membrane cells, the results showed that the number of HEC-1-A cells in each group of Ishikawa cell membrane cells increased significantly, the difference was statistically significant (P0.05). Conclusion: 1 endometrial carcinoma Ishikawa and H The expression of.2 small molecule RNA interference technique can effectively inhibit the expression of ARTN gene at the level of mRNA and protein in ec-1-a cells. The overexpression technology can enhance the expression of ARTN gene and interfere with the expression of ARTN gene, which can inhibit the proliferation activity of endometrial cancer cells and inhibit the migration and invasion ability of the endometrial cancer cells. Overexpression can significantly enhance these effects..HEC-1-A cells have stronger invasion and migration ability than Ishikawa cells.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
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