【摘要】：目的：本實(shí)驗(yàn)以人大腸腺癌SW620細(xì)胞株為研究對象,在細(xì)胞水平上,探討重組質(zhì)粒hARDl-shRNA對5--Fu誘導(dǎo)人大腸腺癌細(xì)胞凋亡的影響,從而為靶向分子治療、靶向分子治療聯(lián)合化療治療大腸癌、研究影響化療敏感性因子提供一些實(shí)驗(yàn)依據(jù),為后續(xù)hARD1的深入研究提供基礎(chǔ)。方法：1、人大腸腺癌細(xì)胞株SW620、shCON-SW620、hARD 1-shRNA-SW620的培養(yǎng)。2、穩(wěn)定轉(zhuǎn)染細(xì)胞系pENTRTM/U6-hARDl-shRNA-SW620的轉(zhuǎn)染率檢測。實(shí)驗(yàn)分組：實(shí)驗(yàn)組：hARDl-shRNA穩(wěn)定轉(zhuǎn)染SW620細(xì)胞。陰性對照組：空載質(zhì)粒轉(zhuǎn)染SW620細(xì)胞。空白對照組：SW620細(xì)胞3、MTT法分別檢測三個實(shí)驗(yàn)組對5-Fu敏感性的影響4、流式細(xì)胞術(shù)Annexin V/PI雙染法檢測細(xì)胞早期凋亡5、流式細(xì)胞術(shù)PI單染法檢測細(xì)胞周期的變化6、Western blot檢測5-Fu作用后的大腸腺癌組織中]hARDl蛋白表達(dá)7, Q-PCR檢測5-Fu作用后的大腸腺癌組織中hARDl mRNA的表達(dá)結(jié)果：1、人大腸腺癌細(xì)胞株SW620、shCON-SW620、hARDl-shRNA-SW620的培養(yǎng)。體外培養(yǎng)成功且生長狀態(tài)良好。2、pENTRTM/U6-hARDl-shRNA-SW620穩(wěn)定轉(zhuǎn)染細(xì)胞系穩(wěn)定性和轉(zhuǎn)染率檢測：shRNA-ARDl SW620培養(yǎng)48h后對hARDl mRNA表達(dá)抑制率達(dá)54.8%。3、MTT法檢測三個實(shí)驗(yàn)組對5-Fu敏感性的影響：分別用不同濃度5-Fu(0、1,、10、20、40、100、500、1000μg/ml)處理各組細(xì)胞觀察各時間段(0、12、24、48、72h)細(xì)胞的生長,測吸光光度值(OD值),計(jì)算腫瘤細(xì)胞抑制率及IC50。SPSS20.0統(tǒng)計(jì)軟件處理,單因素方差分析結(jié)果顯示：瘤細(xì)胞抑制率及IC50實(shí)驗(yàn)組與陰性組、實(shí)驗(yàn)組與空白組比較有統(tǒng)計(jì)學(xué)意義(P0.05)；空白組與陰性對照組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。半數(shù)抑制濃度(IC50)分別為：實(shí)驗(yàn)組344.953±28.539μg/ml,陰性組798.383±86.59μg/ml,空白組735.338±36.530μg/ml4、流式細(xì)胞術(shù)Annexin V/PI雙染法檢測細(xì)胞早期凋亡：無藥物處理的三組細(xì)胞凋亡率比較：實(shí)驗(yàn)組、陰性對照組、空白組無明顯差異,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。低濃度5-Fu(100μg/ml)處理后細(xì)胞凋亡率比較：實(shí)驗(yàn)組較空白組和陰性組細(xì)胞凋亡率增加,有統(tǒng)計(jì)學(xué)差異；高濃度5-Fu(500μg/ml)處理后,細(xì)胞凋亡率比較：實(shí)驗(yàn)組較空白組和陰性組細(xì)胞凋亡率增加,有統(tǒng)計(jì)學(xué)差異(P0.05),空白組與陰性組比較無統(tǒng)計(jì)學(xué)意義(P0.05)。5、流式細(xì)胞術(shù)PI單染法檢測細(xì)胞周期的變化：在5-Fu無藥物作用下,實(shí)驗(yàn)組的G0/G1期細(xì)胞比例較陰性組、空白組高(p0.05)。低濃度(100ug/m1)的作用后各組細(xì)胞的GO/G1期細(xì)胞比例增高,其中實(shí)驗(yàn)組G0/G1期的細(xì)胞比例較陰性組和空白組低高(p0.05)。在5-Fu高濃度(500ug/m1)作用下出現(xiàn)Sub-Gl峰,實(shí)驗(yàn)組G0/G1期細(xì)胞較無藥物組下降(p0.05),但實(shí)驗(yàn)組與陰性對照組和空白對照組無統(tǒng)計(jì)學(xué)差異(p0.05),其細(xì)胞比例較低濃度及無藥物作用組明顯增高(p0.05)。出現(xiàn)Sub-Gl峰,此為亞G1峰,其出現(xiàn)Sub-Gl期細(xì)胞比例較無5-Fu處理和低濃度5-Fu處理時增高,實(shí)驗(yàn)組增高程度較陰性組和空白組高,差異具有統(tǒng)計(jì)學(xué)意義p0.05)。6、Western blot檢測5-Fu作用后的大腸腺癌細(xì)胞中hARD1蛋白表達(dá)：對三組細(xì)胞中hARDl蛋白相對表達(dá)量進(jìn)行灰度值分析,結(jié)果表明：實(shí)驗(yàn)組與對照組兩兩對比差異有統(tǒng)計(jì)學(xué)意義(P0.05)；空白組與陰性對照組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。7、Q-PCR檢測5-Fu作用后的大腸腺癌細(xì)胞中hARDl mRNA的表達(dá)的結(jié)果表明：hARDl mRNA表達(dá)水平,實(shí)驗(yàn)組與陰性組、實(shí)驗(yàn)組與空白組比較有統(tǒng)計(jì)學(xué)意義(P0.05),空白組與陰性對照組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：1. shRNA沉默ARD1基因能提高5-Fu在大腸腺癌SW620細(xì)胞中治療的敏感性,具有化療增敏作用。2. shRNA沉默hARD1基因能促進(jìn)由化療藥物5-Fu引起的大腸癌SW620細(xì)胞的凋亡。3. shRNA沉默ARD1基因聯(lián)合低濃度5-Fu使大腸癌SW620細(xì)胞周期阻滯在G0/G1期,聯(lián)合高濃度5-Fu則促進(jìn)細(xì)胞凋亡。4. shRNA沉默ARD1基因聯(lián)合5-Fu可下調(diào)大腸腺癌細(xì)胞中hARDl蛋白的表達(dá)水平。5. shRNA沉默ARD1基因聯(lián)合5-Fu可下調(diào)大腸腺癌細(xì)胞中hARDlmRNA表達(dá)水平
[Abstract]:Objective: To study the effect of recombinant plasmid hARDl-shRNA on the apoptosis of human colorectal adenocarcinoma cells induced by 5--Fu on the cell level of human colorectal adenocarcinoma cell line SW620, and to provide some experimental evidence for the treatment of colorectal cancer with targeted molecular therapy combined with chemotherapy and the influence of chemotherapy sensitivity factors. In order to provide the basis for further study of hARD1, methods: 1, SW620, shCON-SW620, hARD 1-shRNA-SW620 of human colorectal adenocarcinoma cell line were cultured.2, and the transfection rate of pENTRTM/U6-hARDl-shRNA-SW620 in cell line was stable. Experimental group: experimental group: hARDl-shRNA stably transfected with SW620 cells. Negative control group: empty plasmid transfected SW620 fine. Cell. Blank control group: SW620 cells 3, MTT method detected the effect of three experimental groups on 5-Fu sensitivity respectively 4, flow cytometry Annexin V/PI double staining method to detect cell early apoptosis 5, flow cytometry PI single staining method to detect cell cycle changes 6, Western blot detected the expression of]hARDl protein in colorectal adenocarcinoma after 5-Fu action 7, Q-PCR detection The expression of hARDl mRNA in colorectal adenocarcinoma after 5-Fu action: 1, the culture of SW620, shCON-SW620, hARDl-shRNA-SW620 in the human colorectal adenocarcinoma cell line. In vitro culture, successful and well grown.2, stability and transfection rate of pENTRTM/U6-hARDl-shRNA-SW620 stable transfection cell line: shRNA-ARDl SW620 culture 48h on hARDl The inhibition rate of expression was 54.8%.3. The effect of three experimental groups on the sensitivity of 5-Fu was detected by MTT method: the growth of each time segment (0,12,24,48,72h) cells were observed with different concentrations of 5-Fu (0,1, 10,20,401005001000 mu g/ml), the absorbance photometric value (o value), the tumor cell inhibition rate and the IC50.SPSS20.0 statistical software were calculated. The results of factor variance analysis showed that the tumor cell inhibition rate and the IC50 test group and the negative group were statistically significant (P0.05), and the difference was not statistically significant (P0.05) compared with the negative control group (P0.05). The median inhibitory concentration (IC50) was 344.953 + 28.539 mu g/ml in the experimental group and 798.383 + 86.59 g/ml in the negative group. The blank group was 735.338 + 36.530 g/ml4, and the flow cytometry Annexin V/PI double staining method was used to detect the early apoptosis of the cells. There was no significant difference in the apoptosis rate between the three groups without drug treatment: the experimental group and the negative control group, the difference was not statistically significant (P0.05). The comparison of the apoptosis rate after the treatment of low concentration 5-Fu (100 u g/ml): the experimental group was more empty. The apoptosis rate of cells in the white and negative groups increased statistically. After the treatment of high concentration of 5-Fu (500 u g/ml), the apoptosis rate of the cells in the experimental group was higher than that in the blank group and the negative group (P0.05). There was no significant difference between the blank group and the negative group (P0.05).5, and the flow cytometry PI single staining method was used to detect the cell cycle. The proportion of G0/G1 cells in the experimental group was higher than that in the negative group (P0.05). The proportion of GO/G1 cells in each group increased after the effect of low concentration (100ug/m1), and the proportion of the cells in the G0/G1 phase of the experimental group was higher than that of the negative group and the blank group (P0.05). The Sub-Gl of the experimental group was higher than the negative group and the blank group (P0.05). Sub-Gl was found under the action of 5-Fu high concentration (500ug/m1). Peak, G0/G1 phase cells in the experimental group were lower than that in the non drug group (P0.05), but there was no statistical difference between the experimental group and the negative control group and the blank control group (P0.05), and the proportion of the cells in the lower concentration and the non drug group increased significantly (P0.05). The Sub-Gl peak appeared, this was the sub G1 peak, and the proportion of the cells in the Sub-Gl phase was less than 5-Fu treatment and low concentration 5-Fu treatment. Shi Zenggao, the increase of the experimental group was higher than that of the negative group and the blank group, the difference was statistically significant P0.05).6, and Western blot was used to detect the expression of hARD1 protein in the colorectal adenocarcinoma cells after 5-Fu: the relative expression of hARDl protein in the three groups of cells was analyzed. The results showed that the difference between the experimental group and the control group 22 was statistically different. The difference between the blank group and the negative control group was not statistically significant (P0.05).7. The results of hARDl mRNA expression in colorectal adenocarcinoma cells after the 5-Fu action of 5-Fu showed that the mRNA expression level of hARDl, the experimental group and the negative group, the experimental group and the blank group were statistically significant (P0.05), the blank group and the negative control group were statistically significant (P0.05). The difference was not statistically significant (P0.05). Conclusion: 1. shRNA silencing ARD1 gene can improve the sensitivity of 5-Fu in SW620 cells of colorectal adenocarcinoma, with chemotherapeutic sensitization,.2. shRNA silencing hARD1 gene can promote the apoptosis of colorectal cancer SW620 cells induced by chemotherapeutic drug 5-Fu, and the low concentration of shRNA SW620 cell cycle of colon cancer is blocked in G0/G1 phase, combined with high concentration of 5-Fu,.4. shRNA silencing ARD1 gene combined 5-Fu can downregulate the expression level of hARDl protein in colorectal adenocarcinoma cells,.5. shRNA silencing ARD1 gene combined 5-Fu can down regulate the expression level of colorectal adenocarcinoma cells
【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】：2015
【分類號】：R735.34

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