本文關(guān)鍵詞：沖擊波輔助甲氨蝶呤誘導(dǎo)人骨肉瘤U2OS細(xì)胞凋亡的研究　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細(xì)胞膜通透性 骨肉瘤 沖擊波 P2X7受體

【摘要】：背景:骨肉瘤是最常見的原發(fā)惡性骨腫瘤。骨肉瘤的治療很困難,預(yù)后也差。近年來研究發(fā)現(xiàn)應(yīng)用大劑量化療藥物可以改善骨肉瘤的治療效果,但是這一方法也可以導(dǎo)致許多副作用的產(chǎn)生。如何減少化療藥物的劑量、優(yōu)化治療效果是目前骨肉瘤患者治療的目標(biāo)。如何平衡化療藥物的劑量減少其副作用并保證治療效果,仍然是骨肉瘤治療的一項(xiàng)挑戰(zhàn)。近年來的研究提示沖擊波可能增加骨肉瘤細(xì)胞對化療藥物的敏感性。在本研究中我們應(yīng)用甲氨蝶呤處理人骨肉瘤U2OS細(xì)胞,實(shí)驗(yàn)組輔助沖擊波,評估細(xì)胞的凋亡程度,明確治療效果并探索其潛在機(jī)制。實(shí)驗(yàn)方法:在本研究中,我們應(yīng)用臺盼藍(lán)染色法確定最佳的沖擊波參數(shù)。通過定性和定量分析法測定人骨肉瘤U2OS細(xì)胞對熒光物質(zhì)熒光黃和鈣黃綠素的攝取。應(yīng)用人甲氨蝶呤ELISA試劑盒和MTT法評估沖擊波輔助甲氨蝶呤處理人骨肉瘤U2OS細(xì)胞的效果。為探索沖擊波輔助甲氨蝶呤治療骨肉瘤的機(jī)制,我們應(yīng)用免疫熒光法檢測人骨肉瘤U2OS細(xì)胞膜表面的P2X7受體,應(yīng)用ATP試劑盒檢測細(xì)胞外ATP濃度。全部實(shí)驗(yàn)數(shù)據(jù)應(yīng)用統(tǒng)計(jì)學(xué)軟件SPSS17.0進(jìn)行處理,組間差異采取t檢驗(yàn)進(jìn)行對比。實(shí)驗(yàn)結(jié)果:當(dāng)沖擊波參數(shù)為7 k V 450次或14 k V 200次時,對人骨肉瘤U2OS細(xì)胞活性產(chǎn)生顯著影響。因此實(shí)驗(yàn)中我們選擇沖擊波參數(shù)為7 k V400次和14 k V 150次。實(shí)驗(yàn)結(jié)果表明沖擊波可促使人骨肉瘤U2OS細(xì)胞攝取熒光黃和鈣黃綠素。沖擊波同樣可顯著增加細(xì)胞對化療藥物甲氨蝶呤的攝入并增加甲氨蝶呤誘導(dǎo)的細(xì)胞凋亡。我們發(fā)現(xiàn)沖擊波的處理可使細(xì)胞外ATP濃度增加,人骨肉瘤U2OS細(xì)胞表面大量表達(dá)P2X7受體,P2X7受體抑制劑KN62可以減少甲氨蝶呤誘導(dǎo)的細(xì)胞凋亡。此外我們還發(fā)現(xiàn)只有當(dāng)ATP濃度≥100μM時才對細(xì)胞活性有顯著影響,而沖擊波誘導(dǎo)的ATP釋放濃度最大值不超過1.6μM,說明ATP/P2X7的相互作用沒有直接導(dǎo)致細(xì)胞凋亡。結(jié)論:我們的研究結(jié)果表明:(1)沖擊波可以使人骨肉瘤U2OS細(xì)胞膜的通透性一過性增加,促使大分子物質(zhì)進(jìn)入細(xì)胞內(nèi);(2)沖擊波可以輔助甲氨蝶呤進(jìn)入人骨肉瘤U2OS細(xì)胞內(nèi)并提高化療效果;(3)人骨肉瘤U2OS細(xì)胞膜表面大量表達(dá)P2X7受體;(4)沖擊波可以通過多種途徑促使人骨肉瘤U2OS細(xì)胞內(nèi)ATP向細(xì)胞外釋放;(5)沖擊波輔助甲氨蝶呤誘導(dǎo)人骨肉瘤U2OS細(xì)胞凋亡的機(jī)制為:沖擊波促使人骨肉瘤U2OS細(xì)胞內(nèi)的ATP流向細(xì)胞外,與P2X7受體結(jié)合使細(xì)胞膜的通透性增加,進(jìn)而使大量甲氨蝶呤進(jìn)入細(xì)胞內(nèi),促使細(xì)胞凋亡。臨床意義:沖擊波可能將成為骨肉瘤治療的一種佐劑。骨肉瘤患者在接受化療后可在病灶局部進(jìn)行沖擊波治療,促使腫瘤細(xì)胞攝取化療藥物,這樣就可以減少化療藥物的用量,減少其副作用同時改善骨肉瘤的治療效果。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R738.1

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