【摘要】：低劑量體外沖擊波（Low-dose Extracorporeal Shock Wave,LDESW）可增加細(xì)胞膜的通透性，進(jìn)而使不容易通過(guò)細(xì)胞膜的物質(zhì)更容易通過(guò)細(xì)胞膜進(jìn)入細(xì)胞內(nèi)。體外實(shí)驗(yàn)顯示：體外沖擊波可促進(jìn)化療藥物進(jìn)入細(xì)胞內(nèi)，促進(jìn)細(xì)胞凋亡。這提示：可用體外沖擊波輔助化療藥物治療骨肉瘤。 因此，我們認(rèn)為：體外沖擊波可通過(guò)改變細(xì)胞膜的通透性，定向增加化療藥物進(jìn)入細(xì)胞內(nèi)的含量，從而達(dá)到降低化療藥物的劑量的同時(shí)提高骨肉瘤治療效果的目的。本研究將為如何增加化療藥物對(duì)骨肉瘤的治療效果，減少化療藥物的使用劑量，增進(jìn)腫瘤靶向治療效果，提供一種新的手段和理論依據(jù)。 實(shí)驗(yàn)?zāi)康难芯康蛣┝矿w外沖擊波協(xié)同甲氨蝶呤治療人骨肉瘤的機(jī)制；研究低劑量體外沖擊波能否增加細(xì)胞膜的通透性，使不容易通過(guò)細(xì)胞膜的化療藥物-甲氨蝶呤進(jìn)入細(xì)胞內(nèi)，增加骨肉瘤的治療效果。 實(shí)驗(yàn)方法本研究通過(guò)體外培養(yǎng)骨肉瘤U2OS細(xì)胞，應(yīng)用不同次數(shù)，電容0.3μF、工作電壓7KV和14KV的LDESW分別作用U2OS細(xì)胞，應(yīng)用臺(tái)盼藍(lán)染色法檢測(cè)LDESW對(duì)細(xì)胞活性的影響，分別測(cè)定出7KV和14KV的LDESW對(duì)U2OS細(xì)胞死亡率沒(méi)有顯著性影響(死亡率P＜5%)并且能最大程度增加細(xì)胞膜通透性的臨界次數(shù)。為了探明沖擊波是否能增加骨肉瘤細(xì)胞膜的通透性，我們選擇不能滲透過(guò)細(xì)胞膜的熒光物質(zhì)鈣黃綠素（calcein）和熒光黃（Lucifer Yellow，LY）溶解在細(xì)胞外液，待沖擊波作用后觀(guān)察并比較實(shí)驗(yàn)組和對(duì)照組細(xì)胞內(nèi)熒光物質(zhì)的量。最后，，測(cè)定沖擊波能否促進(jìn)MTX進(jìn)入細(xì)胞內(nèi)，在實(shí)驗(yàn)組與對(duì)照組細(xì)胞懸液內(nèi)加入等濃度的MTX，用沖擊波沖擊后用甲氨蝶呤試劑盒測(cè)定實(shí)驗(yàn)組與對(duì)照組細(xì)胞內(nèi)MTX的濃度，并進(jìn)行比較。 實(shí)驗(yàn)結(jié)果本實(shí)驗(yàn)研究結(jié)果顯示：電容0.3μF、工作電壓7KV的沖擊波作用U2OS細(xì)胞低于400次,電容0.3μF、工作電壓14KV的沖擊波作用U2OS細(xì)胞低于150次對(duì)細(xì)胞死亡率無(wú)顯著性影響（死亡率P＜5%）；加有熒光物質(zhì)的骨肉瘤細(xì)胞懸液用LDESW作用后在熒光顯微鏡下定性觀(guān)察同時(shí)利用熒光/化學(xué)發(fā)光分析儀進(jìn)行定量分析發(fā)現(xiàn)實(shí)驗(yàn)組比對(duì)照組內(nèi)的熒光量明顯增多；用甲氨蝶呤試劑盒測(cè)定經(jīng)過(guò)LDESW作用后的骨肉瘤細(xì)胞內(nèi)的MTX量比對(duì)照組顯著增多。 結(jié)論本研究證實(shí)對(duì)細(xì)胞死亡率無(wú)明顯影響的LDESW能夠使骨肉瘤U2OS細(xì)胞膜通透性一過(guò)性增大，使不容易通過(guò)細(xì)胞膜的MTX更多的進(jìn)入細(xì)胞內(nèi)，起到輔助化療的作用。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R738.1

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