本文關(guān)鍵詞： hARD1 大腸癌 基因沉默 細(xì)胞凋亡因子　出處：《昆明醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]人停滯缺陷蛋白1(human arrest defective 1,hARD1)是酵母N-乙酷基轉(zhuǎn)移酶ARD1的人類同源基因,它與NATI基因結(jié)合,生成NatA復(fù)合體,催化細(xì)胞內(nèi)多種蛋白質(zhì)乙酰化。大腸癌的發(fā)生發(fā)展與hARDI基因表達(dá)及細(xì)胞凋亡密切相關(guān),但hARD1基因表達(dá)能否通過影響細(xì)胞凋亡途徑進(jìn)而影響大腸癌的發(fā)生發(fā)展及具體分子機(jī)制尚不明確。本實驗以人大腸癌SW620細(xì)胞為研究對象,研究hARD1與細(xì)胞凋亡途徑相關(guān)因子之間的關(guān)系,探討hARD1基因被沉默后對凋亡信號通路蛋白的影響,進(jìn)而揭示hARD1基因調(diào)控大腸癌細(xì)胞凋亡具體的分子機(jī)制,以期為大腸癌的早期診斷及治療提供理論依據(jù)。[方法]1.人大腸癌SW620細(xì)胞株的復(fù)蘇、培養(yǎng)、傳代、凍存;2.實驗組即hARD1沉默組,用ARD1 siRNA通過Lipofectamin2000轉(zhuǎn)染試劑轉(zhuǎn)染細(xì)胞株;陰性對照組即hARD1沉默陰性對照組,用NCsiRNA轉(zhuǎn)染細(xì)胞株;空白對照組細(xì)胞株常規(guī)培養(yǎng),不予特殊處理;3. RT-PCR檢測各組細(xì)胞中hARDl的沉默效率;4.流式細(xì)胞術(shù)+PI染色檢測各組大腸癌細(xì)胞的周期變化情況;5.流式細(xì)胞術(shù)和AnnexinV+PI染色檢測各組大腸癌細(xì)胞的凋亡狀況;6.用EdU法檢測各組大腸癌細(xì)胞的增殖狀況;7.熒光探針法檢測各組大腸癌細(xì)胞中ROS含量;8.熒光定量PCR檢測各組大腸癌細(xì)胞中細(xì)胞凋亡途徑相關(guān)因子(TNF-α、Fas、caspase8、Apaf-1、caspase9; Bcl-2、Bad)的mRNA表達(dá)水平;9. Western blot檢測各組大腸癌細(xì)胞中細(xì)胞凋亡途徑相關(guān)因子(TNF-α、Fas、caspase8、Apaf-1、caspase9; Bcl-2、Bad)的蛋白質(zhì)表達(dá)水平;[結(jié)果]1. ARD1siRNA和NCsiRNA轉(zhuǎn)染人大腸癌SW620細(xì)胞后,用RT-PCR檢測各組中hARD1的沉默效率:陰性對照組沉默效率為0%,實驗組的沉默效率為62%;2.流式細(xì)胞術(shù)和AnnexinV+PI染色檢測各組大腸癌細(xì)胞的周期及凋亡情況,結(jié)果顯示:沉默人大腸癌SW620細(xì)胞hARDI基因后,三組細(xì)胞的細(xì)胞周期無明顯改變;實驗組細(xì)胞凋亡水平明顯增加,而陰性對照組和空白對照組無明顯差異;3. EdU檢測細(xì)胞增殖提示:實驗組與空白對照組及陰性對照組比較,實驗組細(xì)胞增殖比例明顯降低,陰性對照組與空白對照組比較細(xì)胞增殖比例無明顯差異;4.熒光探針法檢測ROS含量提示:實驗組與空白對照組及陰性對照組比較,實驗組ROS含量明顯升高,陰性對照組與空白對照組比較ROS含量無明顯差異;5.熒光定量PCR檢測各凋亡因子mRNA表達(dá)水平結(jié)果提示:實驗組的細(xì)胞凋亡途徑相關(guān)因子(caspase9)的mRNA表達(dá)水平明顯高于空白對照組和陰性對照組,而三組中細(xì)胞凋亡途徑相關(guān)因子(TNF-α、caspase8、Fas、Apaf-1、Bcl-2、Bad)的mRNA表達(dá)水平無明顯改變;6. Western blot檢測各凋亡因子蛋白質(zhì)表達(dá)水平結(jié)果提示:實驗組的細(xì)胞凋亡途徑相關(guān)因子(caspase8、caspase9、Fas、Apaf-1、Bcl-2)的蛋白質(zhì)表達(dá)水平明顯高于空白對照組和陰性對照組,而三組中細(xì)胞凋亡途徑相關(guān)因子(TNF-α、Bad)的蛋白質(zhì)表達(dá)水平無明顯改變。[結(jié)論]1.沉默人大腸癌SW620細(xì)胞中hARD1基因后,對其細(xì)胞周期無明顯影響,但能明顯促進(jìn)細(xì)胞凋亡和抑制細(xì)胞增殖;2.本實驗證實,沉默人大腸癌SW620細(xì)胞中hARD1基因后,能夠通過調(diào)節(jié)細(xì)胞凋亡途徑中某些因子的轉(zhuǎn)錄水平和翻譯水平進(jìn)而促進(jìn)細(xì)胞凋亡;3. hARD1基因可能作為大腸癌發(fā)生發(fā)展的潛在作用靶點,為敲除hARD1基因治療大腸癌提供理論依據(jù)。
[Abstract]:[Objective] people Stagnation (human deficient protein 1 defective 1 arrest, hARD1) is the yeast N- B cool glycosyltransferase ARD1 homologous gene of human, it is combined with the NATI gene, generating NatA complex, catalytic intracellular protein acetylation. Colorectal cancer development and hARDI gene expression and cell apoptosis are closely related. But the expression of hARD1 gene can affect the occurrence and development of the molecular mechanism of colorectal cancer by influencing apoptosis pathway is not clear. In this experiment, human colon cancer SW620 cells as the research object, the research of hARD1 and cell apoptosis pathway related with the relationship between, to explore the influence of apoptosis signal pathway protein hARD1 gene silencing, and to reveal the molecular mechanism of hARD1 gene regulation in apoptosis of colorectal cancer cell specific, in order to early diagnosis and treatment of colorectal cancer and provide a theoretical basis. Methods]1. of human colon cancer SW620 cell line. Recovery, culture, subculture, cryopreservation; 2. experimental group hARD1 group ARD1 by siRNA silencing, Lipofectamin2000 transfection reagent transfected cell lines; negative control group: hARD1 silencing negative control group, transfected with NCsiRNA cell line; blank control group cells cultured, not special treatment; hARDl was detected by RT-PCR in 3. the silencing efficiency; cycle changes were detected in colorectal cancer cells 4. +PI staining flow cytometry; apoptosis of colorectal cancer cells were detected by flow cytometry and AnnexinV+PI 5. staining; 6. with EdU method to detect colorectal cancer cell proliferation in colorectal cancer cells; ROS levels were detected in 7. fluorescence probe method; cell apoptosis of colorectal cancer cells were detected by fluorescence quantitative PCR in 8. related factors (Fas, caspase8, TNF- alpha, Apaf-1, caspase9; Bcl-2, Bad) the expression level of mRNA; 9. Western blot were detected in colorectal carcinoma The apoptotic pathway in cell associated factor (TNF- alpha, Fas, caspase8, Apaf-1, caspase9; Bcl-2, Bad) the protein expression of]1. and NCsiRNA ARD1siRNA; the transfection of human colon cancer SW620 cells, using hARD1 RT-PCR to detect the silencing efficiency: negative control group silencing efficiency was 0%, the experimental group silencing efficiency 62%; 2. flow cytometry and AnnexinV+PI staining cycle and apoptosis of colorectal cancer cells were detected, the results showed that the silencing of human colon cancer SW620 cells after hARDI gene, cell cycle of cells in three groups had no obvious change; the level of apoptosis in experimental group increased significantly, while the negative control group and blank control group had no significant difference cell proliferation assay; 3. EdU note: the experimental group and blank control group and negative control group, cell proliferation in experimental group decreased significantly, negative control group and blank control group cell proliferation ratio of ignorance It is suggested that the detection of significant difference; the content of ROS 4. fluorescence probe method: experimental group and blank control group and negative control group, the experimental group was significantly increased ROS content, negative control group and blank control group ROS content had no significant difference; 5. fluorescence quantitative PCR detection of apoptosis factor mRNA expression level showed that cell apoptosis in experimental group the related factor (caspase9) expression level of mRNA was significantly higher than the control group and negative control group, while the apoptosis pathway related factor in the three groups (TNF- alpha, caspase8, Fas, Apaf-1, Bcl-2, Bad) mRNA expression level had no obvious change; 6. Western blot to detect the protein expression of apoptosis factor results the experimental group: cell apoptosis related factors (caspase8, caspase9, Fas, Apaf-1, Bcl-2) protein expression level was significantly higher than that of blank control group and negative control group, while the apoptosis pathway related in three groups Factor (TNF- alpha, Bad) protein expression level had no obvious change in hARD1. Conclusion]1. silencing of human colorectal cancer cell line SW620 in genes that had no significant effect on the cell cycle, but it can significantly promote cell apoptosis and inhibit cell proliferation; 2. confirmed this experiment, hARD1 human colon cancer cells SW620 gene silence, through some factors regulating cell apoptosis pathway in the level of transcription and translation and promote cell apoptosis; potential target gene hARD1 3. may occur as the development of colorectal cancer, to provide theoretical basis for the knockout of hARD1 gene therapy for colorectal cancer.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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本文編號：1512457