DLC-3的表达对MCF-7细胞增殖、迁移及凋亡的影响
发布时间：2018-07-21 15:49
【摘要】：目的:DLC(Deleted in liver cancer)家族基因是新近发现抑癌基因家族,研究证实该家族基因在多种肿瘤组织的低表达或表达缺失。DLCs蛋白在多种肿瘤的发生发展中可能发挥重要作用。本研究旨在探讨DLC-3对乳腺癌MCF-7细胞的增殖、迁移及凋亡生物学行为的影响,观察DLC-3在肿瘤发生和发展的过程中可能起到的作用。方法:(1)通过脂质体搭载目的基因DLC-3,进行瞬时转染使得MCF-7细胞中的DLC-3表达水平显著提高,得到过表达组细胞样本;以相同条件对MCF-7细胞进行空白质粒的转染,得到阴性对照组细胞样本;以常规培养的MCF-7细胞作为实验的空白对照组。转染操作后8h进行换液,常规培养48h后,于荧光显微镜下观察荧光蛋白表达情况,收集各样本细胞用于进一步实验(2)利用Real-time PCR技术对收集的各组样本细胞进行DLC-3的表达水平检测,以阴性对照组ΔCt值为矫正,2-ΔΔCt法进行相对定量分析。(3)CCK-8法检测各样本MCF-7细胞的增殖水平;进行细胞划痕实验观察各样本MCF-7细胞迁移能力的差异;利用流式细胞术进行各样本MCF-7细胞中凋亡细胞比例的比较。结果:(1)过表达组1、2中的DLC-3相对表达量2-ΔΔCt值与空白组比较均存在显著差异(p0.01),瞬时转染后过表达组样本细胞中的DLC-3表达水平显著升高;(2)CCK-8法检测得到:过表达组MCF-7细胞的增殖水平在培养12h、24h、48h后均低于阴性对照组和空白组样本水平,比较显示有显著差异(p0.01),阴性对照组和空白组样本的增殖水平不存在有统计学意义差异(3)细胞划痕实验显示:划痕处理后12h,过表达组样本的愈合率与空白组比较存在显著差异(p0.01),过表达组与阴性对照组,阴性对照与空白组之间愈合率比较差异无统计学意义;划痕处理48h后,过表达组样本的愈合率与阴性对照组以及空白组样本比较均存在显著差异(p0.01),阴性对照组与空白组样本的愈合率比较不存在有统计学意义的差异(4)流式细胞术检测得到:过表达组样本中凋亡细胞所占比率均高于阴性对照组和空白组样本(p0.05),阴性对照组和空白组样本凋亡细胞比率之间不存在差异(p0.05)。结论:通过现象学实验发现乳腺癌MCF-7细胞中DLC-3表达水平提高可以降低细胞增殖水平,抑制其细胞的迁移能力,可以促进其细胞发生凋亡。
[Abstract]:Objective\ *\ The purpose of this study was to investigate the effects of DLC-3 on the biological behavior of proliferation, migration and apoptosis of breast cancer MCF-7 cells, and to observe the possible role of DLC-3 in the development of breast cancer. Methods: (1) transient transfection of the target gene DLC-3 by liposome increased the expression level of DLC-3 in MCF-7 cells, obtained samples of over-expressed cells, and transfected MCF-7 cells with blank plasmids under the same conditions. Cell samples of negative control group were obtained, and MCF-7 cells were cultured as blank control group. After 8 hours of transfection and 48 hours of conventional culture, the expression of fluorescent protein was observed under fluorescence microscope. Each sample cell was collected for further experiment (2) Real-time PCR technique was used to detect the expression of DLC-3 in each group of cells. The relative quantitative analysis was performed with 螖 Ct value of negative control group as correction 2- 螖 Ct method. (3) the proliferation of MCF-7 cells in each sample was detected by CCK-8 method, and the migration ability of MCF-7 cells in each sample was observed by cell scratch test. Flow cytometry was used to compare the percentage of apoptotic cells in MCF-7 cells. Results: (1) there was significant difference in the relative expression of DLC-3 between the overexpression group and the blank group (p0.01), and the expression of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). (2) the results of CCK-8 method showed that: MCF-7 cells in the over-expressed group were detected by CCK-8 method, and the expression level of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). The level of proliferation in the control group and the blank group was lower than that in the negative control group and the blank group after 12 h culture for 24 h or 48 h. Comparison showed significant difference (p0.01). There was no significant difference in proliferative level between negative control group and blank group. (3) Cell scratch test showed that the healing rate of overexpression group was higher than that of blank group 12 hours after scratch treatment. In the significant difference (p0.01), the overexpression group and the negative control group, There was no significant difference in the healing rate between the negative control group and the blank group. There was significant difference in the healing rate between the overexpression group and the negative control group and the blank group (p0.01). There was no significant difference in the healing rate between the negative control group and the blank group (4) flow cytometry was used to detect the healing rate. The results showed that the percentage of apoptotic cells in overexpression group was higher than that in negative control group and blank group (p0.05), but there was no difference between negative control group and blank group (p0.05). Conclusion: the increase of DLC-3 expression in breast cancer MCF-7 cells can reduce the proliferation of MCF-7 cells, inhibit the migration ability of MCF-7 cells and promote the apoptosis of MCF-7 cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2
本文编号：2136009
[Abstract]:Objective\ *\ The purpose of this study was to investigate the effects of DLC-3 on the biological behavior of proliferation, migration and apoptosis of breast cancer MCF-7 cells, and to observe the possible role of DLC-3 in the development of breast cancer. Methods: (1) transient transfection of the target gene DLC-3 by liposome increased the expression level of DLC-3 in MCF-7 cells, obtained samples of over-expressed cells, and transfected MCF-7 cells with blank plasmids under the same conditions. Cell samples of negative control group were obtained, and MCF-7 cells were cultured as blank control group. After 8 hours of transfection and 48 hours of conventional culture, the expression of fluorescent protein was observed under fluorescence microscope. Each sample cell was collected for further experiment (2) Real-time PCR technique was used to detect the expression of DLC-3 in each group of cells. The relative quantitative analysis was performed with 螖 Ct value of negative control group as correction 2- 螖 Ct method. (3) the proliferation of MCF-7 cells in each sample was detected by CCK-8 method, and the migration ability of MCF-7 cells in each sample was observed by cell scratch test. Flow cytometry was used to compare the percentage of apoptotic cells in MCF-7 cells. Results: (1) there was significant difference in the relative expression of DLC-3 between the overexpression group and the blank group (p0.01), and the expression of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). (2) the results of CCK-8 method showed that: MCF-7 cells in the over-expressed group were detected by CCK-8 method, and the expression level of DLC-3 in the overexpression group was significantly higher than that in the control group (p0.01). The level of proliferation in the control group and the blank group was lower than that in the negative control group and the blank group after 12 h culture for 24 h or 48 h. Comparison showed significant difference (p0.01). There was no significant difference in proliferative level between negative control group and blank group. (3) Cell scratch test showed that the healing rate of overexpression group was higher than that of blank group 12 hours after scratch treatment. In the significant difference (p0.01), the overexpression group and the negative control group, There was no significant difference in the healing rate between the negative control group and the blank group. There was significant difference in the healing rate between the overexpression group and the negative control group and the blank group (p0.01). There was no significant difference in the healing rate between the negative control group and the blank group (4) flow cytometry was used to detect the healing rate. The results showed that the percentage of apoptotic cells in overexpression group was higher than that in negative control group and blank group (p0.05), but there was no difference between negative control group and blank group (p0.05). Conclusion: the increase of DLC-3 expression in breast cancer MCF-7 cells can reduce the proliferation of MCF-7 cells, inhibit the migration ability of MCF-7 cells and promote the apoptosis of MCF-7 cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2
【参考文献】
相关期刊论文 前10条
1 陈万青;郑荣寿;;中国女性乳腺癌发病死亡和生存状况[J];中国肿瘤临床;2015年13期
2 陈万青;郑荣寿;曾红梅;邹小农;张思维;赫捷;;2011年中国恶性肿瘤发病和死亡分析[J];中国肿瘤;2015年01期
3 禚守荣;米慧;;乳癌组织肝癌缺失基因-3的表达及其临床意义[J];齐鲁医学杂志;2014年04期
4 李文娟;潘庆杰;李美玉;;p53基因及其功能研究进展[J];生物技术通讯;2014年02期
5 郑莹;吴春晓;张敏璐;;乳腺癌在中国的流行状况和疾病特征[J];中国癌症杂志;2013年08期
6 杨丽丽;梁莉萍;赵峰;;ER、PR、HER-2、Ki67、Nm23、P53在多中心性乳腺癌中的表达及临床意义[J];重庆医科大学学报;2012年11期
7 张敏璐;黄哲宙;郑莹;;中国2008年女性乳腺癌发病、死亡和患病情况的估计及预测[J];中华流行病学杂志;2012年10期
8 郑默然;俸瑞发;柏璐;;血管内皮生长因子(VEGF)在乳腺癌中的研究进展[J];中国医药指南;2012年25期
9 王娜;王玉珍;万永青;谢基明;康鸿斌;刘春霞;;Rab7负向调控巨噬细胞中CpG诱导的细胞因子的表达[J];中国免疫学杂志;2012年08期
10 王红钢;林波;汪文利;;不同转染条件影响质粒转染效率的探讨[J];河南大学学报(医学版);2012年02期
相关硕士学位论文 前1条
1 王剑;SLC22A18过表达对人乳腺癌MCF-7细胞化疗药物敏感性的影响[D];复旦大学;2009年
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