基因RUNX3对肺癌细胞株A549体外作用的研究

发布时间：2018-08-01 09:28

【摘要】：目的:本研究采用质粒载体介导的人类RUNT相关转录因子3(runt related transcription factor 3)在体外通过脂质体感染肺癌细胞株A549,并检测基因对肺癌细胞的增殖、迁移、凋亡的作用。探索基因RUNX3对肺癌增殖、迁移、凋亡的影响,探究潜在的靶向治疗肺癌新靶点。方法:(1)本次实验对四个分组的A549细胞株进行研究,四个分组分别设置为实验组、阳性对照组、阴性对照组和空白组,实验组中加入过表达的Runx3质粒和脂质体Lipofectamine?2000;阳性对照组中加入空载质粒和脂质体Lipofectamine?2000;阴性对照组中加入脂质体Lipofectamine?2000;空白组中只有1640培养基;(2)将过表达RUNX3(EGFP-RUNX3)和质粒(EGFP-NC)扩增提取;(3)通过脂质体Lipofectamine?2000转染过表达RUNX3基因的质粒载体(eGFP-RUNX3)并为实验组、Lipofectamine?2000转染空载质粒(eGFP-NC)为设为阳性对照组、单加Lipofectamine?2000为阴性对照组、单加无血清培养基1640为空白对照组进行实验;(4)使用QPCR检测不同组别的RUNX3 mRNA表达量;(5)使用CCK-8试剂盒检测不同组别的细胞增殖情况;(6)应用Transwell小室实验检测不同组别的细胞株迁移侵袭情况;(7)使用流式细胞学检测不同组别的细胞凋亡情况。结果:(1)本次研究成功提取到过表达Runx3基因质粒和空载质粒,并成功转染到细胞株;(2)荧光定量PCR(QPCR)技术检测结果显示实验组Runx3基因的mRNA表达量最高,显著高于各个对照组(P0.01);(3)CCK-8法分别测定(12h 24h 48h 72h 96h)各组的细胞增殖,结果表明各种细胞随着时间的推移均呈现上升趋势,在增殖实验中转染后72小时实验组过表达RUNX3 OD值(0.883±0.15),阳性对照组(1.638±0.17),阴性对照组(1.631±0.12),空白组(1.788±0.18),实验组明显低于各个对照组(P0.01);(4)侵袭实验中在200倍高倍镜下随机选取三个视野统计平均细胞数量,实验组,阳性对照组,阴性对照组,空白组分别为(28±3.33),(100±2.66),(110±2.33),(180±3.66),实验组明显低于各个对照组(P0.01);(5)Annexin V/PI双染色法检测各组细胞凋亡及坏死比例,结果示实验组、阳性对照组、阴性对照组和空白组的细胞凋亡及坏死比例依次分别为16.25%、5.39%、5.152%、4.202%,实验组的细胞凋亡坏死比例明显高于各个对照组(P0.01)。结论:本实验证实过表达Runx3基因体外抑制肺癌细胞增殖,阻滞肺癌细胞侵袭迁移,导致肺癌细胞凋亡,本研究为分子靶向治疗肺提供了一个新思路和可能新的分子治疗靶点。
[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 卤0.15), (1.638 卤0.17), (1.631 卤0.12) in positive control group, (1.631 卤0.12) in negative control group, and (1.788 卤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 卤3.33), (100 卤2.66), (110 卤2.33), (180 卤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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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