冲击波辅助甲氨蝶呤诱导人骨肉瘤U2OS细胞凋亡的研究

发布时间：2017-12-28 11:08

本文关键词：冲击波辅助甲氨蝶呤诱导人骨肉瘤U2OS细胞凋亡的研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:骨肉瘤是最常见的原发恶性骨肿瘤。骨肉瘤的治疗很困难,预后也差。近年来研究发现应用大剂量化疗药物可以改善骨肉瘤的治疗效果,但是这一方法也可以导致许多副作用的产生。如何减少化疗药物的剂量、优化治疗效果是目前骨肉瘤患者治疗的目标。如何平衡化疗药物的剂量减少其副作用并保证治疗效果,仍然是骨肉瘤治疗的一项挑战。近年来的研究提示冲击波可能增加骨肉瘤细胞对化疗药物的敏感性。在本研究中我们应用甲氨蝶呤处理人骨肉瘤U2OS细胞,实验组辅助冲击波,评估细胞的凋亡程度,明确治疗效果并探索其潜在机制。实验方法:在本研究中,我们应用台盼蓝染色法确定最佳的冲击波参数。通过定性和定量分析法测定人骨肉瘤U2OS细胞对荧光物质荧光黄和钙黄绿素的摄取。应用人甲氨蝶呤ELISA试剂盒和MTT法评估冲击波辅助甲氨蝶呤处理人骨肉瘤U2OS细胞的效果。为探索冲击波辅助甲氨蝶呤治疗骨肉瘤的机制,我们应用免疫荧光法检测人骨肉瘤U2OS细胞膜表面的P2X7受体,应用ATP试剂盒检测细胞外ATP浓度。全部实验数据应用统计学软件SPSS17.0进行处理,组间差异采取t检验进行对比。实验结果:当冲击波参数为7 k V 450次或14 k V 200次时,对人骨肉瘤U2OS细胞活性产生显著影响。因此实验中我们选择冲击波参数为7 k V400次和14 k V 150次。实验结果表明冲击波可促使人骨肉瘤U2OS细胞摄取荧光黄和钙黄绿素。冲击波同样可显著增加细胞对化疗药物甲氨蝶呤的摄入并增加甲氨蝶呤诱导的细胞凋亡。我们发现冲击波的处理可使细胞外ATP浓度增加,人骨肉瘤U2OS细胞表面大量表达P2X7受体,P2X7受体抑制剂KN62可以减少甲氨蝶呤诱导的细胞凋亡。此外我们还发现只有当ATP浓度≥100μM时才对细胞活性有显著影响,而冲击波诱导的ATP释放浓度最大值不超过1.6μM,说明ATP/P2X7的相互作用没有直接导致细胞凋亡。结论:我们的研究结果表明:(1)冲击波可以使人骨肉瘤U2OS细胞膜的通透性一过性增加,促使大分子物质进入细胞内;(2)冲击波可以辅助甲氨蝶呤进入人骨肉瘤U2OS细胞内并提高化疗效果;(3)人骨肉瘤U2OS细胞膜表面大量表达P2X7受体;(4)冲击波可以通过多种途径促使人骨肉瘤U2OS细胞内ATP向细胞外释放;(5)冲击波辅助甲氨蝶呤诱导人骨肉瘤U2OS细胞凋亡的机制为:冲击波促使人骨肉瘤U2OS细胞内的ATP流向细胞外,与P2X7受体结合使细胞膜的通透性增加,进而使大量甲氨蝶呤进入细胞内,促使细胞凋亡。临床意义:冲击波可能将成为骨肉瘤治疗的一种佐剂。骨肉瘤患者在接受化疗后可在病灶局部进行冲击波治疗,促使肿瘤细胞摄取化疗药物,这样就可以减少化疗药物的用量,减少其副作用同时改善骨肉瘤的治疗效果。
[Abstract]:Background: osteosarcoma is the most common primary malignant bone tumor. The treatment of osteosarcoma is difficult and the prognosis is poor. Recent studies have found that large doses of chemotherapeutic drugs can improve the therapeutic effect of osteosarcoma, but this method can also lead to many side effects. How to reduce the dose of chemotherapeutic drugs and optimize the effect of treatment is the target of the treatment of osteosarcoma patients. How to balance the dose of chemotherapeutic drugs to reduce the side effects and ensure the effectiveness of the treatment remains a challenge for the treatment of osteosarcoma. Recent studies have suggested that shock waves may increase the sensitivity of osteosarcoma cells to chemotherapeutic drugs. In this study, methotrexate was used to treat human osteosarcoma U2OS cells. The experimental group was assisted by shock wave to evaluate the degree of apoptosis, and to clarify the therapeutic effect and explore its potential mechanism. Experimental methods: in this study, we used trypan blue to determine the best shock wave parameters. The uptake of fluorescent yellow and calcein in human osteosarcoma U2OS cells was determined by qualitative and quantitative analysis. The effect of impact wave assisted methotrexate on human osteosarcoma U2OS cells was evaluated by methotrexate ELISA kit and MTT. In order to explore the mechanism of shock wave assisted methotrexate in the treatment of osteosarcoma, we detected the P2X7 receptor on the surface of human osteosarcoma U2OS cell by immunofluorescence, and detected the extracellular ATP concentration by ATP kit. All the experimental data were treated with statistical software SPSS17.0, and the difference between groups was compared by t test. The results showed that when the shock wave parameters were 7 K V 450 times or 14 K V 200 times, the activity of U2OS cells in human osteosarcoma was significantly affected. Therefore, we choose the shock wave parameters of 7 K V400 times and 14 K V 150 times. The experimental results show that the shock wave can promote human osteosarcoma U2OS cells and uptake of calcein fluorescent yellow. Shock waves also significantly increase the uptake of methotrexate to chemotherapeutic drugs and increase the apoptosis induced by methotrexate. We found that shock wave treatment can increase extracellular ATP concentration, and P2X7 receptor is expressed on the surface of human osteosarcoma U2OS cells. P2X7 receptor inhibitor KN62 can reduce methotrexate induced apoptosis. In addition, we also found that only when the ATP concentration is 100 M when the cell activity has a significant impact, and the shock wave induced ATP release the maximum concentration of not more than 1.6 M, which indicates that the interaction of ATP/P2X7 does not directly cause cell apoptosis. Conclusion: Our results showed that: (1) shock wave can make the human osteosarcoma U2OS cell membrane permeability had increased, prompting large molecules into cells; (2) the shock wave can be assisted into methotrexate in human osteosarcoma U2OS cells and enhance the effect of chemotherapy; (3) human osteosarcoma cell membrane U2OS surface expression of P2X7 receptor; (4) shock wave through a variety of ways to promote the human osteosarcoma U2OS cells to the extracellular release of ATP; (5) the mechanism of shock wave assisted methotrexate induced apoptosis of human osteosarcoma cell line U2OS: shock wave to make ATP to osteosarcoma cells U2OS cells, combined with with the P2X7 receptor to the cell membrane permeability increased, which causes a large amount of methotrexate into cells, promote cell apoptosis. Clinical significance: shock waves may be an adjuvant for the treatment of osteosarcoma. After receiving chemotherapy, the patients with osteosarcoma can receive shock wave treatment in local area, so that tumor cells can take chemotherapeutic drugs, so that they can reduce the dosage of chemotherapy drugs, reduce their side effects and improve the therapeutic effect of osteosarcoma.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1

【相似文献】