【摘要】：背景與目的:骨肉瘤是臨床上常見的惡性腫瘤,目前其治療方式主要是手術(shù)聯(lián)合藥物化療。然而傳統(tǒng)化療藥物副作用多,對患者二次傷害巨大,因此尋找低毒的新型天然抗癌藥物具有重大意義。早期研究發(fā)現(xiàn)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,參與細胞生長發(fā)育各個過程。目前研究表明MEJA可以抑制多種腫瘤細胞增殖,誘導其凋亡。因此本實驗擬采用MEJA聯(lián)合順鉑(Cisplatin,CDDP)來初步探討其對人骨肉瘤MG-63細胞增殖抑制作用及作用機制。方法:(1)實驗分4個組:空白對照組、MEJA組、CDDP組和MEJA + CDDP藥物聯(lián)合組;(2)采用不同濃度梯度MEJA與CDDP單獨及聯(lián)合作用于MG-63細胞,倒置相差顯微鏡下觀察細胞形態(tài)學變化;(3) Hoechst 33342核染色法及AnnexinV-FITC/Pl流式細胞術(shù)檢測細胞凋亡率;(4) RT-PCR 檢測 Caspase-3、Bcl-2mRNA 表達情況;Western blotting 檢測cleaved Caspase-3、Bcl-2在蛋白水平的表達變化。結(jié)果:(1)顯微鏡下見對照組腫瘤細胞生長良好,而各濃度梯度藥物組細胞則表現(xiàn)為不同程度的凋亡狀態(tài),可見部分細胞脫落降解,貼壁細胞密度降低,部分脫落細胞形態(tài)由梭形變圓,變小,細胞間距增大,胞內(nèi)顆粒增多,鏡下見同一濃度梯度中兩單藥組凋亡細胞數(shù)量大致相同,聯(lián)合組較單藥組凋亡細胞數(shù)量更為顯著,且上述現(xiàn)象隨著藥物濃度的增加而更加明顯;(2) Hoechst 33342核染細胞凋亡熒光檢測結(jié)果發(fā)現(xiàn):對照組中,腫瘤細胞呈均勻的淺藍色熒光,而單藥組中細胞核呈致密濃染的凋亡特征的腫瘤細胞數(shù)量增多,細胞核染成碎塊狀強白光,聯(lián)合組效果更顯著;(3)流式細胞術(shù)檢測結(jié)果顯示:對照組腫瘤細胞凋亡率為8.18± 1.24%,MEJA組腫瘤細胞凋亡率為27.98±2.76%,CDDP組腫瘤細胞凋亡率為31.99土3.6%,MEJA+CDDP藥物聯(lián)合組腫瘤細胞凋亡率為45.77 ±2.76%。兩單藥組腫瘤細胞凋亡率較對照組高,而聯(lián)合組腫瘤細胞凋亡率明顯高于單藥組及對照組;(4) RT-PCR及Western blotting結(jié)果表明:藥物組較對照組而言,Bcl-2表達量下降而Caspase-3表達量增加,聯(lián)合組這一現(xiàn)象更為顯著。結(jié)論:MEJA聯(lián)合CDDP可以協(xié)同抑制MG-63細胞增殖并誘導其凋亡,而MEJA可能是通過下調(diào)Bcl-2及上調(diào)Caspase-3的表達,實現(xiàn)誘導骨肉瘤MG-63細胞生長抑制和凋亡發(fā)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 鹵2.760.The apoptotic rate of tumor cells in the control group was 8.18 鹵1.24 and the apoptotic rate in CDDP group was 31.99 鹵3.6. The apoptotic rate of tumor cells in MEJA CDDP combination group was 45.77 鹵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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R738

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