高良姜诱导肾癌细胞凋亡和抑制细胞侵袭的实验研究

发布时间：2018-10-31 08:31

【摘要】：[目的]：本课题旨在研究高良姜对肾癌786-0和caki-1细胞的体外抗癌作用,同时对高良姜的抗癌作用机制进行初步研究,为高良姜应用于临床治疗肾癌提供一定的实验及理论依据。[方法]：采用CCK-8法分析高良姜对肾癌786-0和caki-1细胞的体外生长抑制作用。采用流式细胞仪分析高良姜诱导肾癌786-0和caki-1细胞凋亡作用。采用相关试剂盒检测肾癌细胞凋亡过程中细胞内SOD(Superoxide dismutase,超氧化物歧化酶)、T-AOC(Total antioxidation,总抗氧化能力)和MDA(Methane Dicarboxylic Aldehyde,丙二醛)的变化,探讨高良姜抗肾癌的ROS(reactive oxygen species,活性氧)相关作用机制。采用划痕实验和Transwell侵袭实验研究高良姜抑制肾癌786-0和caki-1细胞迁移和侵袭的作用。采用western blot(免疫印迹试验)方法研究EMT (Epithelial-Mesenchymal Transition,上皮间质转化)相关蛋白E-cadherin、N-cadherin和vimentin变化,探讨高良姜抑制肾癌细胞侵袭的作用机制。[结果]：1.CCK-8实验结果表明,高良姜对肾癌786-0和caki-1细胞均具有一定的增殖抑制作用,抑制作用随高良姜浓度的增加而增强,高良姜对于786-0和caki-1细胞增殖抑制作用呈浓度依赖性。两种肾癌细胞增殖实验结果均具有显著统计学差异。2.流式细胞仪检测结果表明,高良姜促进肾癌786-0和caki-1细胞的凋亡。与对照组相比,两种肾癌细胞的凋亡均显著升高,凋亡率随高良姜浓度增加呈浓度依赖性增长。实验结果表明,与对照组相比,肾癌786-0和caki-1细胞内抗氧化酶SOD活性随高良姜浓度的增加而显著下降。细胞内T-AOC的活性显著降低,MDA活性显著增加。3.细胞划痕实验和Transwell侵袭实验结果表明,高良姜对于肾癌786-0和caki-1细胞的迁移和侵袭具有抑制作用。划痕试验表明,与对照组相比,100μM高良姜处理组可以显著抑制786-0和caki-1细胞向划痕区域迁移的速度。侵袭实验表明,l00gM高良姜处理组可以显著抑制786-0和caki-1细胞侵袭能力。Western blot实验结果表明,高良姜浓度与细胞内N-cadherin和vimentin蛋白质表达成负相关,与E-cadherin表达成正相关。随高良姜浓度的增加,N-cadherin和vimentin逐渐下降,而E-cadherin逐渐升高。[结论]：1.高良姜具有抑制肾癌786-0和caki-1细胞增殖的作用,抑制作用有明显的浓度依赖性,随浓度上升抑制作用显著增强。2.高良姜具有诱导肾癌786-0和caki-1细胞凋亡的作用。抑制作用有明显的浓度依赖性,随浓度上升抑制作用显著增强。3.高良姜具有抑制肾癌786-0和caki-1细胞迁移和侵袭的作用。4.高良姜通过产生细胞内活性氧诱导肾癌细胞凋亡,并通过抑制细胞间质化抑制肾癌细胞迁移和侵袭。
[Abstract]:[objective]: the aim of this study was to study the anticancer effect of alpinia officinalis on renal cell line 786-0 and caki-1 cells in vitro, and to study the mechanism of anti-cancer effect of alpinia officinalis. To provide a certain experimental and theoretical basis for the clinical treatment of renal cell carcinoma. Methods: CCK-8 assay was used to analyze the growth inhibitory effect of alpinia officinalis on RCC 786-0 and caki-1 cells in vitro. Apoptosis of RCC 786-0 and caki-1 cells was analyzed by flow cytometry. The changes of SOD (Superoxide dismutase, superoxide dismutase (SOD (Superoxide dismutase,), total antioxidant capacity of T-AOC (Total antioxidation, and MDA (Methane Dicarboxylic Aldehyde, malondialdehyde (MDA (Methane Dicarboxylic Aldehyde,) during apoptosis of renal cell carcinoma were detected by relative kit. Objective: to investigate the mechanism of ROS (reactive oxygen species, reactive oxygen species (Ros) related to RCC. The effects of alpinia officinalis on the migration and invasion of renal carcinoma 786-0 and caki-1 cells were studied by scratch test and Transwell invasion assay. The changes of E-cadherin- N-cadherin and vimentin associated proteins of EMT (Epithelial-Mesenchymal Transition, stromal transformation) were studied by western blot (Western blotting assay, and the mechanism of Gastrodia officinalis inhibiting the invasion of renal cancer cells was investigated. [results]: the results of 1.CCK-8 assay showed that alpinia officinalis could inhibit the proliferation of 786-0 and caki-1 cells, and the inhibitory effect increased with the increase of Ginger concentration. The inhibitory effect of alpinia officinale on the proliferation of 786-0 and caki-1 cells was in a concentration dependent manner. There was significant difference between the two kinds of renal cell proliferation test results. 2. 2. The results of flow cytometry showed that alpinia officinalis could promote apoptosis of 786-0 and caki-1 cells. Compared with the control group, the apoptosis rate of the two kinds of renal cancer cells increased in a concentration-dependent manner with the increase of the concentration of alpinia officinalis. The results showed that the activities of antioxidant enzyme SOD in 786-0 and caki-1 cells decreased significantly with the increase of ginger concentration. The activity of T-AOC decreased significantly and the activity of MDA increased significantly. The results of cell scratch assay and Transwell invasion assay showed that alpinia officinalis could inhibit the migration and invasion of RCC 786-0 and caki-1 cells. The scratch test showed that the migration of 786-0 and caki-1 cells to the scratch area was significantly inhibited in the 100 渭 M alpinia officinale treatment group compared with the control group. Invasion experiment showed that l00gM could significantly inhibit the invasiveness of 786-0 and caki-1 cells. The results showed that the concentration of Ginger was negatively correlated with the expression of N-cadherin and vimentin protein in the cells. There was a positive correlation with the expression of E-cadherin. With the increase of ginger concentration, N-cadherin and vimentin decreased gradually, while E-cadherin gradually increased. [conclusion]: 1. Alpinia officinalis has the effect of inhibiting the proliferation of 786-0 and caki-1 cells in a dose-dependent manner, and the inhibitory effect is significantly increased with the increase of concentration. 2. Alpinia officinalis can induce apoptosis of 786-0 and caki-1 cells in renal carcinoma. The inhibitory effect was obviously concentration-dependent, and the inhibitory effect was significantly increased with the increase of concentration. Alpinia officinalis can inhibit the migration and invasion of 786-0 and caki-1 cells. 4. Ginger induces apoptosis of renal cell carcinoma cells by producing intracellular reactive oxygen species, and inhibits migration and invasion of renal cancer cells by inhibiting interstitial cells.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11

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