体外冲击波辅助甲氨蝶呤治疗骨肉瘤的基础研究

发布时间：2018-08-31 19:06

【摘要】：低剂量体外冲击波（Low-dose Extracorporeal Shock Wave,LDESW）可增加细胞膜的通透性，进而使不容易通过细胞膜的物质更容易通过细胞膜进入细胞内。体外实验显示：体外冲击波可促进化疗药物进入细胞内，促进细胞凋亡。这提示：可用体外冲击波辅助化疗药物治疗骨肉瘤。因此，我们认为：体外冲击波可通过改变细胞膜的通透性，定向增加化疗药物进入细胞内的含量，从而达到降低化疗药物的剂量的同时提高骨肉瘤治疗效果的目的。本研究将为如何增加化疗药物对骨肉瘤的治疗效果，减少化疗药物的使用剂量，增进肿瘤靶向治疗效果，提供一种新的手段和理论依据。实验目的研究低剂量体外冲击波协同甲氨蝶呤治疗人骨肉瘤的机制；研究低剂量体外冲击波能否增加细胞膜的通透性，使不容易通过细胞膜的化疗药物-甲氨蝶呤进入细胞内，增加骨肉瘤的治疗效果。实验方法本研究通过体外培养骨肉瘤U2OS细胞，应用不同次数，电容0.3μF、工作电压7KV和14KV的LDESW分别作用U2OS细胞，应用台盼蓝染色法检测LDESW对细胞活性的影响，分别测定出7KV和14KV的LDESW对U2OS细胞死亡率没有显著性影响(死亡率P＜5%)并且能最大程度增加细胞膜通透性的临界次数。为了探明冲击波是否能增加骨肉瘤细胞膜的通透性，我们选择不能渗透过细胞膜的荧光物质钙黄绿素（calcein）和荧光黄（Lucifer Yellow，LY）溶解在细胞外液，待冲击波作用后观察并比较实验组和对照组细胞内荧光物质的量。最后，，测定冲击波能否促进MTX进入细胞内，在实验组与对照组细胞悬液内加入等浓度的MTX，用冲击波冲击后用甲氨蝶呤试剂盒测定实验组与对照组细胞内MTX的浓度，并进行比较。实验结果本实验研究结果显示：电容0.3μF、工作电压7KV的冲击波作用U2OS细胞低于400次,电容0.3μF、工作电压14KV的冲击波作用U2OS细胞低于150次对细胞死亡率无显著性影响（死亡率P＜5%）；加有荧光物质的骨肉瘤细胞悬液用LDESW作用后在荧光显微镜下定性观察同时利用荧光/化学发光分析仪进行定量分析发现实验组比对照组内的荧光量明显增多；用甲氨蝶呤试剂盒测定经过LDESW作用后的骨肉瘤细胞内的MTX量比对照组显著增多。结论本研究证实对细胞死亡率无明显影响的LDESW能够使骨肉瘤U2OS细胞膜通透性一过性增大，使不容易通过细胞膜的MTX更多的进入细胞内，起到辅助化疗的作用。
[Abstract]:Low dose extracorporeal shock wave (Low-dose Extracorporeal Shock Wave,LDESW) can increase the permeability of cell membrane and make it easier for substances not easily passed through the cell membrane to enter into the cell membrane. In vitro experiments showed that extracorporeal shock wave could promote chemotherapeutic drugs into cells and promote apoptosis. This suggests that extracorporeal shock wave chemotherapeutic drugs can be used to treat osteosarcoma. Therefore, we think that the extracorporeal shock wave can increase the content of chemotherapeutic drugs by changing the permeability of cell membrane, so as to reduce the dose of chemotherapeutic drugs and improve the therapeutic effect of osteosarcoma. This study will provide a new method and theoretical basis for increasing the therapeutic effect of chemotherapeutic drugs on osteosarcoma, reducing the dosage of chemotherapeutic drugs, and improving the therapeutic effect of tumor targeting. Objective to study the mechanism of low dose extracorporeal shock wave combined with methotrexate in the treatment of human osteosarcoma, and whether the low dose extracorporeal shock wave can increase the permeability of cell membrane and make it difficult to enter the cell through the chemotherapeutic drug methotrexate. To increase the therapeutic effect of osteosarcoma. Methods Osteosarcoma U2OS cells were cultured in vitro. U2OS cells were treated with LDESW of 0.3 渭 F capacitance, working voltage 7KV and 14KV, respectively. The effect of LDESW on cell activity was detected by trypan blue staining. The LDESW of 7KV and 14KV had no significant effect on the cell death rate of U2OS (P < 5%) and could increase the critical number of cell membrane permeability. In order to find out whether shock wave can increase the permeability of osteosarcoma cell membrane, we choose the fluorescent substances that can not penetrate the cell membrane, calcium xanthophyllin (calcein) and fluorescent yellow (Lucifer Yellow,LY) dissolved in the extracellular fluid. After shock wave action, the amount of intracellular fluorescence was observed and compared between the experimental group and the control group. Finally, whether shock wave could promote MTX into the cells was determined. The concentration of MTX in the cells of experimental group and control group was measured by methotrexate kit after the same concentration of MTX, was added into the cell suspension of experimental group and control group. Results the experimental results showed that the cell death rate was not significantly affected by the shock wave action of 0.3 渭 F, the shock wave of working voltage 7KV was less than 400 times, the capacitance was 0.3 渭 F, and the shock wave of working voltage 14KV was less than 150 times (P < 5%). After the suspension of osteosarcoma cells with fluorescent substance was treated with LDESW, the quantity of fluorescence in the experimental group was significantly higher than that in the control group after qualitative observation under fluorescence microscope and quantitative analysis by fluorescence / chemiluminescence analyzer. The amount of MTX in osteosarcoma cells treated with LDESW was significantly increased with methotrexate kit. Conclusion LDESW, which has no obvious effect on cell death rate, can make the permeability of U2OS cell membrane increase temporarily, which makes it difficult to enter the cells through the MTX of the cell membrane and play the role of adjuvant chemotherapy.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1

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