茉莉酸甲酯联合顺铂抑制人骨肉瘤MG-63细胞增殖作用的研究
发布时间：2018-12-12 01:44
【摘要】：背景与目的:骨肉瘤是临床上常见的恶性肿瘤,目前其治疗方式主要是手术联合药物化疗。然而传统化疗药物副作用多,对患者二次伤害巨大,因此寻找低毒的新型天然抗癌药物具有重大意义。早期研究发现茉莉酸甲酯(Methyl Jasmonate,MEJA)在植物界普遍存在且具有重要生理功能,参与细胞生长发育各个过程。目前研究表明MEJA可以抑制多种肿瘤细胞增殖,诱导其凋亡。因此本实验拟采用MEJA联合顺铂(Cisplatin,CDDP)来初步探讨其对人骨肉瘤MG-63细胞增殖抑制作用及作用机制。方法:(1)实验分4个组:空白对照组、MEJA组、CDDP组和MEJA + CDDP药物联合组;(2)采用不同浓度梯度MEJA与CDDP单独及联合作用于MG-63细胞,倒置相差显微镜下观察细胞形态学变化;(3) Hoechst 33342核染色法及AnnexinV-FITC/Pl流式细胞术检测细胞凋亡率;(4) RT-PCR 检测 Caspase-3、Bcl-2mRNA 表达情况;Western blotting 检测cleaved Caspase-3、Bcl-2在蛋白水平的表达变化。结果:(1)显微镜下见对照组肿瘤细胞生长良好,而各浓度梯度药物组细胞则表现为不同程度的凋亡状态,可见部分细胞脱落降解,贴壁细胞密度降低,部分脱落细胞形态由梭形变圆,变小,细胞间距增大,胞内颗粒增多,镜下见同一浓度梯度中两单药组凋亡细胞数量大致相同,联合组较单药组凋亡细胞数量更为显著,且上述现象随着药物浓度的增加而更加明显;(2) Hoechst 33342核染细胞凋亡荧光检测结果发现:对照组中,肿瘤细胞呈均匀的浅蓝色荧光,而单药组中细胞核呈致密浓染的凋亡特征的肿瘤细胞数量增多,细胞核染成碎块状强白光,联合组效果更显著;(3)流式细胞术检测结果显示:对照组肿瘤细胞凋亡率为8.18± 1.24%,MEJA组肿瘤细胞凋亡率为27.98±2.76%,CDDP组肿瘤细胞凋亡率为31.99土3.6%,MEJA+CDDP药物联合组肿瘤细胞凋亡率为45.77 ±2.76%。两单药组肿瘤细胞凋亡率较对照组高,而联合组肿瘤细胞凋亡率明显高于单药组及对照组;(4) RT-PCR及Western blotting结果表明:药物组较对照组而言,Bcl-2表达量下降而Caspase-3表达量增加,联合组这一现象更为显著。结论:MEJA联合CDDP可以协同抑制MG-63细胞增殖并诱导其凋亡,而MEJA可能是通过下调Bcl-2及上调Caspase-3的表达,实现诱导骨肉瘤MG-63细胞生长抑制和凋亡发生。
[Abstract]:Background & objective: osteosarcoma is a common malignant tumor. However, traditional chemotherapeutic drugs have many side effects and great secondary injury to patients, so it is of great significance to find new natural anticancer drugs with low toxicity. Early studies have found that methyl jasmonate (Methyl Jasmonate,MEJA) exists widely in plant and has important physiological function, which is involved in various processes of cell growth and development. Current studies have shown that MEJA can inhibit the proliferation and induce apoptosis of various tumor cells. In this study, MEJA combined with cisplatin (Cisplatin,CDDP) was used to investigate the inhibitory effect of MEJA on the proliferation of human osteosarcoma MG-63 cells and its mechanism. Methods: (1) the experiment was divided into four groups: blank control group, MEJA group, CDDP group and MEJA CDDP combination group; (2) MG-63 cells were treated with different concentration gradient MEJA and CDDP alone or in combination, and the morphologic changes of MG-63 cells were observed by inverted phase contrast microscope. (3) Hoechst 33342 nuclear staining and AnnexinV-FITC/Pl flow cytometry were used to detect the apoptosis rate of MG-63 cells. (4) the expression of Caspase-3,Bcl-2mRNA was detected by RT-PCR.; Western blotting was used to detect the expression of cleaved Caspase-3,Bcl-2 at protein level. Results: (1) under microscope, the tumor cells in the control group grew well, while the cells in each concentration gradient drug group showed different degree of apoptosis, and some of the cells fell off and degraded, and the density of adherent cells decreased. The number of apoptotic cells in the same concentration gradient was approximately the same, and the number of apoptotic cells in the combination group was more significant than that in the single drug group, and the number of apoptotic cells in the combined group was higher than that in the single drug group. The above phenomenon is more obvious with the increase of drug concentration. (2) the results of Hoechst 33342 nuclear staining showed that: in the control group, the tumor cells showed light blue fluorescence, while in the single drug group, the number of tumor cells with dense and dense staining was increased. The nucleus was dyed into pieces of strong white light, and the effect of the combined group was more obvious than that of the control group. (3) the results of flow cytometry showed that the apoptotic rate of tumor cells in MEJA group was 27.98 卤2.760.The apoptotic rate of tumor cells in the control group was 8.18 卤1.24 and the apoptotic rate in CDDP group was 31.99 卤3.6. The apoptotic rate of tumor cells in MEJA CDDP combination group was 45.77 卤2.76. The apoptosis rate of tumor cells in the two single drug groups was higher than that in the control group, while the apoptosis rate in the combined group was significantly higher than that in the single drug group and the control group. (4) the results of RT-PCR and Western blotting showed that the expression of Bcl-2 decreased and the expression of Caspase-3 increased in the drug group compared with the control group, which was more significant in the combined group. Conclusion: MEJA combined with CDDP can synergistically inhibit the proliferation and induce apoptosis of MG-63 cells, while MEJA may induce the growth inhibition and apoptosis of osteosarcoma MG-63 cells by down-regulating Bcl-2 and upregulating the expression of Caspase-3.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738
[Abstract]:Background & objective: osteosarcoma is a common malignant tumor. However, traditional chemotherapeutic drugs have many side effects and great secondary injury to patients, so it is of great significance to find new natural anticancer drugs with low toxicity. Early studies have found that methyl jasmonate (Methyl Jasmonate,MEJA) exists widely in plant and has important physiological function, which is involved in various processes of cell growth and development. Current studies have shown that MEJA can inhibit the proliferation and induce apoptosis of various tumor cells. In this study, MEJA combined with cisplatin (Cisplatin,CDDP) was used to investigate the inhibitory effect of MEJA on the proliferation of human osteosarcoma MG-63 cells and its mechanism. Methods: (1) the experiment was divided into four groups: blank control group, MEJA group, CDDP group and MEJA CDDP combination group; (2) MG-63 cells were treated with different concentration gradient MEJA and CDDP alone or in combination, and the morphologic changes of MG-63 cells were observed by inverted phase contrast microscope. (3) Hoechst 33342 nuclear staining and AnnexinV-FITC/Pl flow cytometry were used to detect the apoptosis rate of MG-63 cells. (4) the expression of Caspase-3,Bcl-2mRNA was detected by RT-PCR.; Western blotting was used to detect the expression of cleaved Caspase-3,Bcl-2 at protein level. Results: (1) under microscope, the tumor cells in the control group grew well, while the cells in each concentration gradient drug group showed different degree of apoptosis, and some of the cells fell off and degraded, and the density of adherent cells decreased. The number of apoptotic cells in the same concentration gradient was approximately the same, and the number of apoptotic cells in the combination group was more significant than that in the single drug group, and the number of apoptotic cells in the combined group was higher than that in the single drug group. The above phenomenon is more obvious with the increase of drug concentration. (2) the results of Hoechst 33342 nuclear staining showed that: in the control group, the tumor cells showed light blue fluorescence, while in the single drug group, the number of tumor cells with dense and dense staining was increased. The nucleus was dyed into pieces of strong white light, and the effect of the combined group was more obvious than that of the control group. (3) the results of flow cytometry showed that the apoptotic rate of tumor cells in MEJA group was 27.98 卤2.760.The apoptotic rate of tumor cells in the control group was 8.18 卤1.24 and the apoptotic rate in CDDP group was 31.99 卤3.6. The apoptotic rate of tumor cells in MEJA CDDP combination group was 45.77 卤2.76. The apoptosis rate of tumor cells in the two single drug groups was higher than that in the control group, while the apoptosis rate in the combined group was significantly higher than that in the single drug group and the control group. (4) the results of RT-PCR and Western blotting showed that the expression of Bcl-2 decreased and the expression of Caspase-3 increased in the drug group compared with the control group, which was more significant in the combined group. Conclusion: MEJA combined with CDDP can synergistically inhibit the proliferation and induce apoptosis of MG-63 cells, while MEJA may induce the growth inhibition and apoptosis of osteosarcoma MG-63 cells by down-regulating Bcl-2 and upregulating the expression of Caspase-3.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738
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