【摘要】：目的:本研究采用質粒載體介導的人類RUNT相關轉錄因子3(runt related transcription factor 3)在體外通過脂質體感染肺癌細胞株A549,并檢測基因對肺癌細胞的增殖、遷移、凋亡的作用。探索基因RUNX3對肺癌增殖、遷移、凋亡的影響,探究潛在的靶向治療肺癌新靶點。方法:(1)本次實驗對四個分組的A549細胞株進行研究,四個分組分別設置為實驗組、陽性對照組、陰性對照組和空白組,實驗組中加入過表達的Runx3質粒和脂質體Lipofectamine?2000;陽性對照組中加入空載質粒和脂質體Lipofectamine?2000;陰性對照組中加入脂質體Lipofectamine?2000;空白組中只有1640培養(yǎng)基;(2)將過表達RUNX3(EGFP-RUNX3)和質粒(EGFP-NC)擴增提取;(3)通過脂質體Lipofectamine?2000轉染過表達RUNX3基因的質粒載體(eGFP-RUNX3)并為實驗組、Lipofectamine?2000轉染空載質粒(eGFP-NC)為設為陽性對照組、單加Lipofectamine?2000為陰性對照組、單加無血清培養(yǎng)基1640為空白對照組進行實驗;(4)使用QPCR檢測不同組別的RUNX3 mRNA表達量;(5)使用CCK-8試劑盒檢測不同組別的細胞增殖情況;(6)應用Transwell小室實驗檢測不同組別的細胞株遷移侵襲情況;(7)使用流式細胞學檢測不同組別的細胞凋亡情況。結果:(1)本次研究成功提取到過表達Runx3基因質粒和空載質粒,并成功轉染到細胞株;(2)熒光定量PCR(QPCR)技術檢測結果顯示實驗組Runx3基因的mRNA表達量最高,顯著高于各個對照組(P0.01);(3)CCK-8法分別測定(12h 24h 48h 72h 96h)各組的細胞增殖,結果表明各種細胞隨著時間的推移均呈現(xiàn)上升趨勢,在增殖實驗中轉染后72小時實驗組過表達RUNX3 OD值(0.883±0.15),陽性對照組(1.638±0.17),陰性對照組(1.631±0.12),空白組(1.788±0.18),實驗組明顯低于各個對照組(P0.01);(4)侵襲實驗中在200倍高倍鏡下隨機選取三個視野統(tǒng)計平均細胞數(shù)量,實驗組,陽性對照組,陰性對照組,空白組分別為(28±3.33),(100±2.66),(110±2.33),(180±3.66),實驗組明顯低于各個對照組(P0.01);(5)Annexin V/PI雙染色法檢測各組細胞凋亡及壞死比例,結果示實驗組、陽性對照組、陰性對照組和空白組的細胞凋亡及壞死比例依次分別為16.25%、5.39%、5.152%、4.202%,實驗組的細胞凋亡壞死比例明顯高于各個對照組(P0.01)。結論:本實驗證實過表達Runx3基因體外抑制肺癌細胞增殖,阻滯肺癌細胞侵襲遷移,導致肺癌細胞凋亡,本研究為分子靶向治療肺提供了一個新思路和可能新的分子治療靶點。
[Abstract]:Aim: to investigate the effect of gene on proliferation, migration and apoptosis of lung cancer cell line A549 in vitro, using plasmid vector mediated human RUNT related transcription factor 3 (runt related transcription factor 3 (3 (runt related transcription factor 3) to infect lung cancer cell line A549 through liposome in vitro. To explore the effects of gene RUNX3 on proliferation, migration and apoptosis of lung cancer, and explore potential new targets for lung cancer therapy. Methods: (1) four A549 cell lines were divided into four groups: experimental group, positive control group, negative control group and blank group. Runx3 plasmid and liposome Lipofectamine2000 were added in the experimental group; empty plasmids and liposome Lipofectamine2000 were added in the positive control group; liposome Lipofectamine 2000 was added in the negative control group; only 1640 medium was added in the blank group; (2) overexpression of RUNX3 (EGFP-RUNX3) and plasmid (EGFP-NC) were amplified. (3) the plasmid vector (eGFP-RUNX3) expressing RUNX3 gene was transfected by liposome Lipofectamine?2000 and the blank plasmid (eGFP-NC) was transfected with Lipofectamine2000 in the experimental group as the positive control group. Lipofectamine?2000 alone was used as negative control group. Single serum-free medium 1640 was used as blank control group; (4) QPCR was used to detect the expression of RUNX3 mRNA in different groups; (5) CCK-8 kit was used to detect cell proliferation in different groups; (6) Transwell chamber experiment was used to detect the expression of RUNX3 mRNA in different groups. Cell line migration and invasion; (7) flow cytometry was used to detect cell apoptosis in different groups. Results: (1) Runx3 gene plasmid and empty plasmid were extracted successfully and transfected into cell line successfully. (2) the results of fluorescence quantitative PCR (QPCR) showed that the mRNA expression of Runx3 gene was the highest in the experimental group. Compared with the control group (P0.01); (3), the cell proliferation was measured by CCK-8 assay (12 h, 24 h, 48 h, 72 h, 96 h), respectively. The results showed that all kinds of cells showed an upward trend with the passage of time. The OD value of RUNX3 was (0.883 鹵0.15), (1.638 鹵0.17), (1.631 鹵0.12) in positive control group, (1.631 鹵0.12) in negative control group, and (1.788 鹵0.18) in blank group at 72 hours after transfection. The OD value of RUNX3 in experimental group was significantly lower than that in each control group (P0.01); (4) invasion experiment. The average number of cells in the field, In the experimental group, the positive control group, the negative control group and the blank group were (28 鹵3.33), (100 鹵2.66), (110 鹵2.33), (180 鹵3.66) respectively. The ratio of apoptosis and necrosis in the experimental group was significantly lower than that in the control group (P0.01); (5) Annexin V/PI double staining. The results showed that the percentage of apoptosis and necrosis in the experimental group was significantly lower than that in the control group (P0.01); (5). The percentages of apoptosis and necrosis in negative control group and blank group were 16.255.395.395.152 and 5.152.The percentage of apoptosis and necrosis in experimental group was significantly higher than that in each control group (P0.01). Conclusion: this study confirmed that overexpression of Runx3 gene can inhibit the proliferation of lung cancer cells, block the invasion and migration of lung cancer cells, and lead to apoptosis of lung cancer cells in vitro. This study provides a new idea and a possible new molecular therapeutic target for molecular targeted therapy of lung cancer.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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