2-CDA对黑色素瘤细胞增殖和凋亡的影响及其机制

发布时间：2018-06-22 19:16

本文选题：恶性黑色素瘤 + 克拉屈滨　；参考：《辽宁医学院》2011年硕士论文

【摘要】：目的恶性黑色素瘤(malignant melanoma ,MM)是所有恶性肿瘤中发病率增长最快的肿瘤,年增长率为3-5%[1]。MM是一种高度恶性的肿瘤,尽管它只占皮肤黏膜肿瘤的4%左右,但是它造成的死亡人数却占到了皮肤黏膜相关癌死亡总人数的80%,而且一旦MM发生转移,病人的5年生存期只有14%[2]。对于早期能够完整切除原发灶的病例采取手术切除,预后良好,但是对于原发灶不能完整切除或者发生转移的病例目前还缺乏有效的药物治疗。所以现在亟需探索新疗法。本文运用体外实验筛选出2-CDA分别在24h、48h时段内杀灭人黑色素瘤A375细胞和小鼠黑色素瘤B16细胞的最佳浓度,同时观察2-CDA对黑色素瘤A375细胞和小鼠黑色素瘤B16细胞的增殖、形态、周期、迁移和凋亡的影响,并探讨其作用机制,为黑色素瘤的临床治疗提供依据。方法 0.4%的台酚蓝计数法观察不同浓度的2-CDA处理B16细胞对其细胞增殖的影响;蛋白印迹(western blot)检测B16细胞内凋亡标志蛋白的变化情况。MTT比色法检测2-CDA处理对A375细胞细胞增殖的影响;瑞氏-姬姆萨染色观察细胞形态的变化;流式细胞仪分析细胞周期的改变;划痕修复实验检测A375细胞迁移能力的变化; Annexin-V/PI双标法检测药物对A375细胞凋亡的诱导作用;蛋白印迹(western blot)检测A375细胞内凋亡标志蛋白的变化情况。结果 2-CDA对B16细胞和A375细胞的增殖抑制随着时间的延长和剂量的增加而增强;瑞氏-姬姆萨染色显示2-CDA处理细胞30 h后,细胞数明显减少,细胞形态不规则,多数细胞体积固缩。流式细胞仪检测表明A375细胞阻滞于S期的比例随2-CDA剂量增加而增加。划痕修复实验结果显示2-CDA能够显著抑制A375细胞的迁移能力;Annexin-V/PI双标法检测结果表明2-CDA可诱导A375细胞凋亡;Western blot检测结果显示,2-CDA处理A375细胞和B16细胞后,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。结论 2-CDA能够抑制B16增殖并诱导B16细胞发生凋亡。2-CDA能够对A375细胞增殖、形态、周期、迁移等生物学性质产生广泛的影响,并能激活Caspase3信号通路诱导A375细胞凋亡。
[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.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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