【摘要】： 皮膚黑素瘤是一種高度惡性的皮膚腫瘤,其發(fā)病機制仍未完全闡明。近來研究發(fā)現(xiàn)腫瘤抑制基因胰島素樣生長因子結(jié)合蛋白7(insulin-like growth factor binding protein 7,IGFBP7)的表達受抑制是黑素瘤發(fā)生的一個重要環(huán)節(jié),抑癌基因的表達異常與DNA異常甲基化相關(guān),本課題的目的是研究皮膚黑素瘤細胞中IGFBP7基因的表達調(diào)控機制,重點闡明啟動子區(qū)CpG島異常甲基化對IGFBP7基因表達調(diào)控的影響。 1 IGFBP7在皮膚黑素瘤組織和人黑素瘤細胞株中的表達 應(yīng)用免疫組化法檢測發(fā)現(xiàn),IGFBP7蛋白在原發(fā)性皮膚黑素瘤和轉(zhuǎn)移性黑素瘤組織中陽性表達率明顯低于普通良性色素痣組織,有顯著性差異。應(yīng)用半定量RT-PCR和免疫細胞化學法檢測發(fā)現(xiàn),IGFBP7mRNA和蛋白在人黑素瘤細胞株A375、M14和SK-MEL-1中均陰性表達,在原代黑素細胞和人黑素瘤細胞株MV3中陽性表達。結(jié)果顯示,IGFBP7在皮膚黑素瘤組織和人黑素瘤細胞株中表達抑制。 2人黑素瘤細胞株IGFBP7基因啟動子區(qū)CpG島異常甲基化的研究 應(yīng)用亞硫酸氫鈉修飾后測序法(bisulfite sequencing PCR, BSP)檢測人黑素瘤細胞株A375、M14、SK-MEL-1、MV3和原代黑素細胞中IGFBP7基因啟動子區(qū)CpG島所有CpG位點的甲基化狀態(tài),結(jié)果顯示,在IGFBP7表達陰性和陽性的細胞之間存在顯著的甲基化差異。 35-aza-dC對人黑素瘤A375、M14細胞IGFBP7的表達及生物學行為的影響 應(yīng)用去甲基化藥物5-aza-dC處理陰性表達IGFBP7的A375和M14細胞,藥物處理后,2株細胞IGFBP7mRNA和蛋白均恢復(fù)表達,且BSP檢測證實了該基因啟動子區(qū)發(fā)生了去甲基化改變,驗證了IGFBP7在這2株細胞中的表達恢復(fù)是該基因去甲基化的直接作用,表明啟動子區(qū)CpG島DNA異常甲基化是黑素瘤細胞IGFBP7表達改變的直接調(diào)控機制。 經(jīng)MTT實驗、流式細胞術(shù)、TUNEL法和trans well小室侵襲實驗檢測發(fā)現(xiàn),5-aza-dC在體外抑制A375和M14細胞增殖,促進凋亡,并導(dǎo)致細胞周期出現(xiàn)G1期阻滯,抑制細胞侵襲。結(jié)合藥物處理后IGFBP7的表達變化,推測IGFBP7表達的恢復(fù),很可能參與了5-aza-dC處理所引起的黑素瘤細胞上述生物學行為的改變。 以上研究結(jié)果說明,IGFBP7基因啟動子區(qū)CpG島DNA異常甲基化是黑素瘤細胞中IGFBP7表達改變的主要調(diào)控機制。去甲基化藥物5-aza-dC還有抑制腫瘤細胞生長、阻滯細胞周期、促進細胞凋亡、抑制腫瘤細胞侵襲的作用,IGFBP7表達的恢復(fù)很可能在5-aza-dC引起的黑素瘤細胞生物學行為改變中發(fā)揮作用。
[Abstract]:Skin melanoma is a highly malignant skin tumor, the pathogenesis of which has not been fully clarified. Recently, it has been found that the inhibition of the expression of tumor inhibitor gene insulin-like growth factor binding protein 7 (insulin-like growth factor binding protein 7, IGFBP7) is an important link in the occurrence of melanoma. The abnormal expression of tumor inhibitor gene is related to the abnormal methylation of DNA. The purpose of this study was to study the regulatory mechanism of IGFBP7 gene expression in skin melanoma cells, and to elucidate the effect of abnormal methylation of CpG island in promoter region on the regulation of IGFBP7 gene expression. 1 the expression of 1 IGFBP7 in skin melanoma tissue and human melanoma cell line was detected by immunohistochemical method, and the results were as follows: (1) the expression of VEGF in skin melanoma tissue and human melanoma cell line was detected by immunohistochemistry. The positive expression rate of IGFBP7 protein in primary skin melanoma and metastatic melanoma was significantly lower than that in normal benign pigmented nevi. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the negative expression of IGFBP7mRNA and protein in human melanoma cell line A375, M14 and SK-MEL-1, and positive expression in primary melanocytes and human melanoma cell line MV3. The results showed that the expression of IGFBP7 was inhibited in skin melanoma tissue and human melanoma cell line. Abnormal methylation of CpG island in promoter region of IGFBP7 gene of two human melanoma cell lines (bisulfite sequencing PCR, BSP) was used to detect human melanoma cell line A375, M14, SK / MEL 鈮，

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