Artemin基因在子宮內(nèi)膜癌細(xì)胞增殖及侵襲中的作用
發(fā)布時(shí)間：2018-08-09 09:05
【摘要】：子宮內(nèi)膜癌是女性生殖系統(tǒng)最常見的三大惡性腫瘤之一,近些年其發(fā)病率有上升趨勢(shì)。大多數(shù)子宮內(nèi)膜癌患者發(fā)病處于早期,經(jīng)外科手術(shù)治療預(yù)后較好;但仍有少數(shù)子宮內(nèi)膜癌患者就診時(shí)已處于疾病晚期,有報(bào)道晚期或經(jīng)化療復(fù)發(fā)患者的中位生存時(shí)間不超過1年。子宮內(nèi)膜癌的發(fā)病類型可分為雌激素依賴型(Ⅰ型)和非雌激素依賴型(Ⅱ型),前者占內(nèi)膜癌大多數(shù),預(yù)后相對(duì)較好;后者因其生物特性具有侵襲性,具有早期轉(zhuǎn)移的傾向,預(yù)后通常較差。Artemin(ARTN)是膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子(gilal cell line derived neurotrophic factor,GDNF)家族中的一個(gè)成員,ARTN基因在體內(nèi)廣泛分布,提示它對(duì)多系統(tǒng)組織、器官的發(fā)育及生理功能的維持具有重要意義。ARTN基因同胰腺癌、食管癌、胃癌、乳腺癌等的發(fā)生、發(fā)展密切相關(guān)。本課題通過干擾及過表達(dá)ARTN基因,進(jìn)一步研究ARTN基因?qū)θ俗訉m內(nèi)膜癌細(xì)胞增殖、遷移及侵襲的影響。目的:從細(xì)胞水平探討干擾及過表達(dá)ARTN基因?qū)θ俗訉m內(nèi)膜癌細(xì)胞株的增殖、遷移及侵襲的影響,通過靶向干擾ARTN基因表達(dá),為子宮內(nèi)膜癌的靶向治療提供一定理論依據(jù)。方法:1體外培養(yǎng)子宮內(nèi)膜癌Ishikawa和HEC-1-A細(xì)胞,應(yīng)用Western Blot方法檢測(cè)兩種細(xì)胞中ARTN蛋白表達(dá)情況。2以pGPU6/GFP/Neo為載體構(gòu)建ARTN基因的短發(fā)夾RNA(pGPU6/GFP/Neo-ARTN shRNA)干擾質(zhì)粒,以pcDNA3.1為克隆載體,構(gòu)建ARTN基因的過表達(dá)質(zhì)粒(pcDNA3.1-ARTN)。3將ARTN基因的干擾及過表達(dá)質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染人子宮內(nèi)膜癌Ishikawa和HEC-1-A細(xì)胞,利用qRT-PCR及Western Blot法檢測(cè)轉(zhuǎn)染后兩種細(xì)胞中ARTN mRNA及蛋白表達(dá)變化。4應(yīng)用CCK法繪制細(xì)胞生長(zhǎng)曲線,檢測(cè)細(xì)胞增殖能力,同時(shí)利用transwell小室實(shí)驗(yàn)檢測(cè)細(xì)胞遷移及侵襲能力變化。結(jié)果:1兩種子宮內(nèi)膜癌細(xì)胞中均有artn蛋白不同程度的表達(dá),相對(duì)表達(dá)量分別為0.808±0.015和0.895±0.008。2轉(zhuǎn)染pgpu6/gfp/neo-artnshrna干擾質(zhì)粒后,兩種細(xì)胞的artnmrna相對(duì)表達(dá)量明顯下降(f=47.882,f=394.326,p0.05);轉(zhuǎn)染pcdna3.1-artn過表達(dá)質(zhì)粒后,兩種細(xì)胞artnmrna相對(duì)表達(dá)量則明顯增加(f=276.007,f=861.159,p0.05),差異有統(tǒng)計(jì)學(xué)意義。干擾artn基因表達(dá)后,兩種細(xì)胞中的artn蛋白相對(duì)表達(dá)量均明顯減少(f=726.323,f=1780.714,p0.05);強(qiáng)制表達(dá)artn基因后,兩種細(xì)胞中的artn蛋白相對(duì)表達(dá)量則明顯增加(f=1145.127,f=519.357,p0.05),差異均有統(tǒng)計(jì)學(xué)意義。3轉(zhuǎn)染artn干擾質(zhì)粒24h,兩種細(xì)胞增殖能力無明顯變化(p0.05),轉(zhuǎn)染48h、72h、96h兩種細(xì)胞增殖能力均明顯降低(p0.05);轉(zhuǎn)染artn過表達(dá)質(zhì)粒24h,兩種細(xì)胞增殖能力無明顯變化(p0.05),轉(zhuǎn)染48h、72h、96h兩種細(xì)胞增殖能力均明顯增強(qiáng)(p0.05)。4侵襲實(shí)驗(yàn)結(jié)果顯示兩種細(xì)胞干擾組比較陰性對(duì)照組和對(duì)照組穿膜細(xì)胞數(shù)明顯減少,差異有統(tǒng)計(jì)學(xué)意義(f=55.609,f=108.865,p0.05);過表達(dá)組比較陰性對(duì)照組和對(duì)照組穿膜細(xì)胞數(shù)則明顯增加,差異有統(tǒng)計(jì)學(xué)意義(f=42.880,f=32.004,p0.05)。遷移實(shí)驗(yàn)中兩種細(xì)胞干擾組與陰性對(duì)照組、對(duì)照組比較,穿膜細(xì)胞數(shù)明顯減少(f=52.938,f=90.224,p0.05);過表達(dá)組與陰性對(duì)照組、對(duì)照組比較,穿膜細(xì)胞數(shù)明顯增加(f=196.899,f=100.167,p0.05),差異均有統(tǒng)計(jì)學(xué)意義;侵襲、遷移實(shí)驗(yàn)中將兩種細(xì)胞的干擾組、過表達(dá)組和對(duì)照組穿膜細(xì)胞數(shù)進(jìn)行對(duì)比,結(jié)果發(fā)現(xiàn)hec-1-a細(xì)胞比較ishikawa細(xì)胞各組穿膜細(xì)胞數(shù)都明顯增加,差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:1子宮內(nèi)膜癌ishikawa和hec-1-a細(xì)胞中均有不同強(qiáng)度的artn蛋白的表達(dá)。2小分子rna干擾技術(shù)可以有效抑制artn基因在mrna及蛋白質(zhì)水平的表達(dá);過表達(dá)技術(shù)則可增強(qiáng)artn基因的表達(dá)。3干擾ARTN基因表達(dá)可顯著抑制子宮內(nèi)膜癌細(xì)胞的增殖活性,抑制其遷移及侵襲能力;過表達(dá)則可顯著增強(qiáng)這些作用。HEC-1-A細(xì)胞比Ishikawa細(xì)胞具有更強(qiáng)的侵襲及遷移能力。
[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.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33
本文編號(hào)：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.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33
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