本文選題：惡性黑色素瘤 + 克拉屈濱�。� 參考：《遼寧醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的 惡性黑色素瘤(malignant melanoma ,MM)是所有惡性腫瘤中發(fā)病率增長最快的腫瘤,年增長率為3-5%[1]。MM是一種高度惡性的腫瘤,盡管它只占皮膚黏膜腫瘤的4%左右,但是它造成的死亡人數(shù)卻占到了皮膚黏膜相關(guān)癌死亡總?cè)藬?shù)的80%,而且一旦MM發(fā)生轉(zhuǎn)移,病人的5年生存期只有14%[2]。對于早期能夠完整切除原發(fā)灶的病例采取手術(shù)切除,預(yù)后良好,但是對于原發(fā)灶不能完整切除或者發(fā)生轉(zhuǎn)移的病例目前還缺乏有效的藥物治療。所以現(xiàn)在亟需探索新療法。本文運(yùn)用體外實(shí)驗(yàn)篩選出2-CDA分別在24h、48h時(shí)段內(nèi)殺滅人黑色素瘤A375細(xì)胞和小鼠黑色素瘤B16細(xì)胞的最佳濃度,同時(shí)觀察2-CDA對黑色素瘤A375細(xì)胞和小鼠黑色素瘤B16細(xì)胞的增殖、形態(tài)、周期、遷移和凋亡的影響,并探討其作用機(jī)制,為黑色素瘤的臨床治療提供依據(jù)。 方法 0.4%的臺酚藍(lán)計(jì)數(shù)法觀察不同濃度的2-CDA處理B16細(xì)胞對其細(xì)胞增殖的影響;蛋白印跡(western blot)檢測B16細(xì)胞內(nèi)凋亡標(biāo)志蛋白的變化情況。MTT比色法檢測2-CDA處理對A375細(xì)胞細(xì)胞增殖的影響;瑞氏-姬姆薩染色觀察細(xì)胞形態(tài)的變化;流式細(xì)胞儀分析細(xì)胞周期的改變;劃痕修復(fù)實(shí)驗(yàn)檢測A375細(xì)胞遷移能力的變化; Annexin-V/PI雙標(biāo)法檢測藥物對A375細(xì)胞凋亡的誘導(dǎo)作用;蛋白印跡(western blot)檢測A375細(xì)胞內(nèi)凋亡標(biāo)志蛋白的變化情況。 結(jié)果 2-CDA對B16細(xì)胞和A375細(xì)胞的增殖抑制隨著時(shí)間的延長和劑量的增加而增強(qiáng);瑞氏-姬姆薩染色顯示2-CDA處理細(xì)胞30 h后,細(xì)胞數(shù)明顯減少,細(xì)胞形態(tài)不規(guī)則,多數(shù)細(xì)胞體積固縮。流式細(xì)胞儀檢測表明A375細(xì)胞阻滯于S期的比例隨2-CDA劑量增加而增加。劃痕修復(fù)實(shí)驗(yàn)結(jié)果顯示2-CDA能夠顯著抑制A375細(xì)胞的遷移能力;Annexin-V/PI雙標(biāo)法檢測結(jié)果表明2-CDA可誘導(dǎo)A375細(xì)胞凋亡;Western blot檢測結(jié)果顯示,2-CDA處理A375細(xì)胞和B16細(xì)胞后,Caspase-3被剪切活化,其底物蛋白聚ADP核糖聚合酶(PARP)也被剪切形成89 kD的剪切片段。 結(jié)論 2-CDA能夠抑制B16增殖并誘導(dǎo)B16細(xì)胞發(fā)生凋亡。2-CDA能夠?qū)375細(xì)胞增殖、形態(tài)、周期、遷移等生物學(xué)性質(zhì)產(chǎn)生廣泛的影響,并能激活Caspase3信號通路誘導(dǎo)A375細(xì)胞凋亡。
[Abstract]:Objective malignant melanoma (malignant melanoma) is the fastest growing tumor of all malignant tumors, with an annual growth rate of 3 to 5% [1] .MM is a highly malignant tumor, although it only accounts for about 4% of skin and mucosal tumors. However, the death toll is 80% of the total death toll of skin and mucosa-associated cancer, and once MM metastases, the patient's 5-year survival rate is only 14% [2]. The prognosis is good for the patients who can remove the primary tumor completely in the early stage. However, there is still no effective drug treatment for the patients with incomplete resection or metastasis of the primary tumor. So there is an urgent need to explore new treatments. The optimal concentrations of 2-CDA for killing human melanoma A375 cells and mouse melanoma B16 cells within 48 hours were screened out by in vitro experiments. The proliferation and morphology of 2-CDA on melanoma A375 cells and mouse melanoma B16 cells were observed at the same time. The effects of cycle, migration and apoptosis, and its mechanism are discussed to provide evidence for the clinical treatment of melanoma. Methods the effects of 2-CDA treatment on the proliferation of B16 cells were observed by 0.4% table-blue counting method. Western blot (western blot) was used to detect the changes of apoptosis marker protein in B16 cells. MTT colorimetric assay was used to detect the effect of 2-CDA treatment on the proliferation of A375 cells. Scratch repair assay was used to detect the migration ability of A375 cells. Annexin-V / Pi double labeling assay was used to detect the apoptosis induction of A375 cells. Western blot (western blot) was used to detect the changes of apoptosis markers in A375 cells. Results the proliferation inhibition of 2-CDA on B16 cells and A375 cells increased with the increase of time and dose, and the number of cells decreased and the cell morphology was irregular after treated with 2-CDA for 30 h. Most cells shrink in volume. Flow cytometry showed that the proportion of A375 cells blocked in S phase increased with the increase of 2-CDA dose. The results of scratch repair test showed that 2-CDA could significantly inhibit the migration ability of A375 cells. The results of Annexin-V / Pi double labeling assay showed that 2-CDA could induce apoptosis of A375 cells. Western blot analysis showed that Caspase-3 was shearing and activated after treated with 2-CDA in A375 cells and B16 cells. Its substrate protein poly (ADP) ribosomal polymerase (PARP) was also cut into 89 KD shear fragments. Conclusion 2-CDA can inhibit the proliferation of B16 and induce apoptosis of B16 cells. 2-CDA can have extensive effects on the biological properties of A375 cells, such as proliferation, morphology, cycle and migration, and can activate Caspase3 signaling pathway to induce apoptosis of A375 cells.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.5

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