本文選題：鎘 + A549細(xì)胞�。� 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：鎘(Cadmium)是一種在環(huán)境中存在十分廣泛的有毒重金屬,普遍應(yīng)用于制造陶瓷釉彩、鎘電池、復(fù)寫紙和照相機及合成纖維印染助劑等。鎘被人體吸收后,會通過鎘硫蛋白的形式蓄積在腎、肝、腦、肺、膀胱、前列腺以及子宮內(nèi)膜中,從而對多個器官系統(tǒng)造成巨大的危害,1993年鎘被國際癌癥研究機構(gòu)列為Ⅰ類致癌物。除了職業(yè)性暴露以外,人們還會通過吸煙和食用被污染的食物這種非職業(yè)性暴露的形式吸收鎘。目前,鎘被發(fā)現(xiàn)可以擾亂DNA修復(fù)系統(tǒng),進(jìn)而促進(jìn)癌癥的形成和發(fā)展,其還可以增強一些組織和細(xì)胞的遷移、侵襲能力。高遷移率族蛋白A2(HMGA2)在真核生物中大量存在,因為分子質(zhì)量小,且在凝膠電泳中遷移率高而得名。其自身缺乏轉(zhuǎn)錄活性,但具有三個特殊的AT-hooks結(jié)構(gòu),這些AT-hooks結(jié)構(gòu)可以與DNA中AT富集序列特異性結(jié)合,從而改變基因的DNA結(jié)構(gòu),或直接作用于相關(guān)基因,加強或者抑制它們的轉(zhuǎn)錄活性,進(jìn)而影響胚胎形成和組織發(fā)育,以及引起腫瘤的發(fā)生。因為HMGA2具有這種通過改變?nèi)旧|(zhì)的結(jié)構(gòu)來調(diào)節(jié)很多靶基因表達(dá)的特點,它常被稱做“構(gòu)架轉(zhuǎn)錄因子”。HMGA2的表達(dá)升高在胚胎形成期幾乎無處不在,并且對于胚胎發(fā)育期細(xì)胞的增長繁殖和分化起了必不可少的作用。它同時被發(fā)現(xiàn)在很多腫瘤中表達(dá)升高。HMGA2的大量表達(dá)與結(jié)腸癌晚期、浸潤型乳腺癌的高組織學(xué)分級,以及乳腺癌、結(jié)腸癌、肺癌患者的低生存率相關(guān)。但是HMGA2是否參與了鎘誘導(dǎo)的遷移和侵襲尚不十分清楚。目的:探討HMGA2在鎘誘導(dǎo)的遷移和侵襲中的作用。方法:本研究以人肺腺癌細(xì)胞(A549細(xì)胞)、人胚腎細(xì)胞(HEK 293細(xì)胞)和小鼠肺組織為研究對象。按實驗設(shè)計,對小鼠連續(xù)6周皮下注射不同濃度鎘(0,0.25,0.5和1mg/kg),提取小鼠肺組織蛋白;將低濃度(2μM)氯化鎘作用于A549細(xì)胞和HMGA2 siRNA處理后的A549細(xì)胞;pc DNA3.1-HMGA2質(zhì)粒轉(zhuǎn)染HEK 293細(xì)胞得到HMGA2表達(dá)升高的細(xì)胞模型。應(yīng)用蛋白印跡法(Western blot)檢測細(xì)胞和組織中HMGA2蛋白和遷移侵襲相關(guān)蛋白MMP-9、MMP-2、FAK以及p-FAK水平的變化。通過Transwell小室實驗觀察低濃度鎘作用后A549細(xì)胞遷移侵襲能力的變化。實驗結(jié)果采用獨立樣本t檢驗進(jìn)行分析。結(jié)果:每天給小鼠皮下注射不同濃度鎘連續(xù)6周后,鎘處理組HMGA2的表達(dá)量與對照組相比明顯增高(P0.01),說明低濃度鎘可以誘導(dǎo)小鼠肺組織中HMGA2的表達(dá)。同時,遷移侵襲相關(guān)蛋白MMP-9、MMP-2和p-FAK的表達(dá)顯著升高(P0.01),提示低濃度鎘可以使小鼠肺組織細(xì)胞的遷移侵襲能力增強。在人肺腺癌細(xì)胞(A549細(xì)胞)中,用低濃度(2μM)氯化鎘染毒48小時后,檢測到HMGA2、MMP-9、MMP-2和p-FAK蛋白表達(dá)量顯著升高(P0.01),表明低濃度鎘可以誘導(dǎo)A549細(xì)胞的遷移侵襲相關(guān)蛋白的表達(dá),Transwell小室實驗中,鎘處理組A549細(xì)胞遷移和侵襲通過孔徑膜和基質(zhì)膠的數(shù)量與對照組相比明顯增高(P0.01)。用HMGA2 siRNA處理A549細(xì)胞使HMGA2表達(dá)下降以后,遷移侵襲相關(guān)蛋白MMP-9、MMP-2和p-FAK的表達(dá)顯著下降(P0.01),并且Transwell小室實驗中用HMGA2 siRNA處理后的染鎘組遷移和侵襲通過孔徑膜和基質(zhì)膠的A549細(xì)胞數(shù)量與control siRNA處理后的染鎘組相比顯著減少(P0.01)。pc DNA3.1-HMGA2質(zhì)粒轉(zhuǎn)染HEK 293細(xì)胞使HMGA2的表達(dá)升高后,遷移侵襲相關(guān)蛋白MMP-9、MMP-2和p-FAK的表達(dá)與陰性對照組相比顯著升高(P0.01)。這些結(jié)果表明HMGA2在鎘誘導(dǎo)的遷移侵襲中發(fā)揮了重要作用。結(jié)論:鎘可以增強小鼠肺組織和A549細(xì)胞的遷移、侵襲能力;HMGA2在鎘誘導(dǎo)的遷移侵襲中發(fā)揮了重要作用。
[Abstract]:Cadmium (Cadmium) is a very widespread toxic heavy metal in the environment. It is widely used in the manufacture of ceramic glaze color, cadmium battery, duplex paper, camera and synthetic fiber printing and dyeing auxiliaries. Cadmium is absorbed by human body and accumulates in the kidney, liver, brain, lung, bladder, prostate and endometrium through the absorption of cadmium sulfide in the form of cadmium sulphide protein. The official system has caused great harm. In 1993, cadmium was classified as a type I carcinogen by the International Cancer Research Institute. In addition to occupational exposure, cadmium can also be absorbed in the form of nonoccupational exposure to smoking and food contaminated food. Cadmium has been found to disrupt the DNA repair system and promote the formation and development of cancer. It can also enhance the migration and invasiveness of some tissues and cells. High mobility group protein A2 (HMGA2) exists in eukaryotes, because of its small molecular weight and high mobility in gel electrophoresis. It lacks transcriptional activity, but has three special AT-hooks structures, and these AT-hooks structures can be enriched with AT in DNA. Let heterosexual binding, which changes the DNA structure of the gene, or directly acts on the related genes, strengthens or inhibits their transcriptional activity, and then affects the formation and development of the embryos, and causes the occurrence of tumors. Because HMGA2 has the characteristics of many target genes by changing the structure of chromatin, it is often called to be called. The expression of "structural transcription factor".HMGA2 is almost ubiquitous in the embryonic period, and is essential for the growth and differentiation of cells in embryonic development. It is also found to be expressed in many tumors and the high expression of.HMGA2 in the late stage of colon cancer and the high histological grade of invasive breast cancer. It is related to the low survival rate of patients with breast cancer, colon cancer and lung cancer. But whether HMGA2 is involved in cadmium induced migration and invasion is not very clear. Objective: To explore the role of HMGA2 in cadmium induced migration and invasion. Methods: This study was conducted with human lung adenocarcinoma cells (A549 cells), human embryonic kidney cells (HEK 293 cells) and mice lung tissue Object. According to the experimental design, mice were subcutaneously injected with different concentrations of cadmium (0,0.25,0.5 and 1mg/kg) for 6 weeks to extract the mouse lung tissue protein; the low concentration (2 M) of cadmium chloride was acted on the A549 cells treated with A549 cells and HMGA2 siRNA; PC DNA3.1-HMGA2 plasmid transfected to HEK 293 cells to obtain the cell model of HMGA2 expression. The changes in the level of HMGA2 protein and migration associated protein MMP-9, MMP-2, FAK and p-FAK in cells and tissues were detected by Western blot. The changes in the migration and invasion of A549 cells after low concentration of cadmium were observed through the Transwell chamber experiment. The experimental results were analyzed by independent sample t examination. Results: the mice were injected subcutaneously every day. After 6 weeks of cadmium concentration, the expression of HMGA2 in the cadmium treatment group was significantly higher than that in the control group (P0.01), indicating that the low concentration of cadmium could induce the expression of HMGA2 in the lung tissue of mice. Meanwhile, the migration and invasion related protein MMP-9, the expression of MMP-2 and p-FAK increased significantly (P0.01), suggesting that low concentration of cadmium could induce migration of lung tissue cells in mice. In human lung adenocarcinoma cells (A549 cells), the expression of HMGA2, MMP-9, MMP-2 and p-FAK was significantly increased (P0.01) after 48 hours of cadmium chloride exposure at low concentration (2 u M), indicating that low concentration of cadmium could induce the expression of migration and invasion of A549 cells, and the migration of A549 cells in the cadmium treatment group in the Transwell lab experiment The number of and invasion through the aperture membrane and matrix gel was significantly higher than that in the control group (P0.01). After the A549 cells were treated with HMGA2 siRNA to decrease the HMGA2 expression, the migration and invasion related protein MMP-9, the expression of MMP-2 and p-FAK decreased significantly (P0.01), and in the Transwell lab experiment, the migration and invasion of the cadmium exposed group after HMGA2 siRNA were passed through the Transwell chamber experiment. The number of A549 cells in the aperture membrane and the matrix gel decreased significantly compared with that of the control siRNA treated group (P0.01). The.Pc DNA3.1-HMGA2 plasmid transfected with HEK 293 cells increased the expression of HMGA2, and the expression of the migration related proteins MMP-9, MMP-2 and p-FAK increased significantly compared with the negative control group (P0.01). These results showed that the cadmium DNA3.1-HMGA2 was induced by cadmium. Conclusion: cadmium can enhance the migration and invasiveness of lung and A549 cells in mice, and HMGA2 plays an important role in the migration and invasion induced by cadmium.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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本文編號：1822750