【作者】 王玉芳；

【导师】 王树人； 周桥；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 研究背景与目的:恶性黑色素瘤是来源于皮肤黑素细胞的一种恶性程度很高的肿瘤,突出特点为发病早,转移率高,致死率高。大量的研究表明恶性黑色素瘤中细胞凋亡的“关闭”导致其对临床各种化疗药物耐受,从而具备了强大的生存能力。近年来,有部分研究报道了与细胞凋亡相关的凋亡调节分子和执行分子在该肿瘤中的表达以及变异情况,虽然局限于某一个分子在恶性黑色素细胞中表达异常,如Apaf-1,survivin,bcl-2等,这些研究结果对于深入认识恶性黑色素瘤耐药的分子机制提供了线索。但是恶性黑色素瘤对多种治疗措施都不敏感就说明该肿瘤的耐药机制很复杂,因此我们以四株恶性黑色素瘤细胞作为研究对象,对与凋亡相关的基因和蛋白质表达情况进行系统的研究,以期从整体的角度来认识该疾病与凋亡的关系,为筛选出可以作为临床诊断和预后的特征性的分子奠定基础。 材料与方法:运用基因芯片技术获得四株恶性黑色素瘤细胞凋亡相关基因表达谱;利用RT-PCR、Western Blot、免疫组织化学和原位杂交技术在恶性黑色素瘤细胞和组织中检测caspase14的表达情况;采用MTT方法测定抗癌药和UV刺激对两大类恶性黑色素瘤细胞增殖活力的影更多还原

【Abstract】 Background and objective: Melanoma is the most aggressive form of skin cancer and notoriously resistant to all current modalities of cancer therapy. A large set of genetic, functional and biochemical studies suggest that melanoma cells become "bullet proof against a variety of chemotherapeutic drugs by reprogramming their proliferation and survival pathways during melanoma progression. In recent years, the identification of molecules involved in the regulation and execution of apoptosis, and their alteration in melanoma, have provided new insights into the molecular basis for melanoma chemoresistance. But the wide range of antineoplastic treatments that are ineffective at killing melanoma cells implies that the resistance mechanism in melanoma are complex. Using gene chip andWestern blot, we have achieved expression profile of apoptosis-related genes and proteins in melanoma cells in order to the discovery of independent predictors of clinical outcome.Material and methods: We applied gene chip to achieve the expression profile of melanoma cells; Both caspasel4 mRNA and protein expression were examined in melanoma cells, melanocytes, melanoma tissue and nevus by RT-PCR, Western Blot, immunohistochemistry (IHC) and hybridization in situ.The proliferation was determined after UV-Light irradiation, camptothecin and cisplatin for indicated times(0> 6> 12,24h). And caspases and related-apoptosis proteins were examined in two kinds of melanoma cells and melanocytes by Western Blot.Results: From expression profile of related-apoptosis gene in melanoma cells:(l) Among Bcl-2 family, BaxNMcl-1 %Bcl-XL -. Bar and Nip3 presented positive signals; (2)signals of c-IAPl and Apollon/Bruce, which belong to IAP family were positively expressed; (3)MDM2 which is oncogene and HUS-1 which is cell cyclin check point gene showed positive signals; (4) the moleculors in TNF receptor family HVEM-1, TNFR2/p75 fP LTbR expressed positively; (5)caspasel4 was identified in both melanoma cells, melanocytes and melanoma tissue and nevus. Both caspaseH mRNA and protein level in cell line A375> M14 and SK-Mel-1, marked by MART-1 high expression, are higher than in cell line A875, marked by MART-1 low expression. CaspaseH protein level were elevated in melanoma tissue in contrast to nevus.Mart-1 high expressed cell lines were more suecepted to UV-Light irradiation and chemotherapeutic drugs camptothecin and cisplatin. We furtherdetected the expression of caspases and related-apoptosis protein between two kinds of melanoma and melanocytes. (l)The levels of anti-apoptosis protein ML-IAL, Bcl-2 were increased in Mart-1 high expressed cell lines in contrast to Mart-1 low expressed cell line and melanocytes; (2) inhibitor of apoptosis protein c-IAPl, C-IAP2 and survivin level were lower in Mart-1 low expressed cell line than Mart-1 high expressed cell lines and melanocytes,but there was no difference between Mart-1 high expressed cell lines and melanocytes; (3)the expression of pro-apoptosis protein SMAC decreased in melanoma cells in contrast to melanocytes;(4) We did not identify the expression of caspase6> 7> 8^ 10; but the level of caspase3 and caspase9 were increased in Mart-1 high expressed cell lines; (5) Apaf-1 protein level was decreased only in cell line Ml4, while it had no difference between other melanoma cell lines and melanocytes; (6) caspase3 protein level unexpectedly increased in Mart-1 high expressed cell line in contrast to melanocytes.Conclusion: From the results of expression profile of related-apoptosis gene, we can conclude : overexpress Mcl-1 and Bcl-XL may be players in overriding the apoptotic signals generated by increased Bax and decreased Bcl-2 expression; c-IAPl and Apollon/Bruce could block the apoptosis by inhibiting caspases activation ; and we discover firstly the other members of Bcl-2 family : Ba^ Nip3 , oncogene MDM2 and cell cyclin check point gene HUS-1 may be taken part in the mechanism of melanoma chemoresistance.We first reported caspasel4 was expressed in both melanoma cells, melanocytes and melanoma tissue and nevus. The level of Caspasel4 mRNA and protein were elevated in melanoma in contrast to nevus.Using UV, camptothecin and cisplatin irradiation, Mart-1 high expressed cell lines were more resistant than Mart-1 low expressed cell lines. It was possible mechanism that the level of anti-apoptosis protein, including ML-IAP, Bcl-2, c-IAPl, c-IAP2and survivin, in Mart-1 high expressed cell lines expressed higher than Mart-1 low expressed cell lines, while the level of pro-apoptosis protein, including caspase3, caspase9, SMAC, in Mart-1 high expressed cell lines expressed lower than Mart-1 low expressed cell lines. These results mignt have implication for clinic prognosis.Between Mart-1 high expressed cell lines and melanocytes, the level of anti-apoptosis protein, including ML-IAP, Bcl-2, c-IAPl, C-IAP2 and survivin were increased in Mart-1 high expressed cell lines; while the level of pro-apoptosis protein SMAC was decreased in Mart-1 high expressed cell lines, these results may be related to melanoma resistant to all anticancer therapies. Caspase3, caspase9 unexpectedly upregulated in Mart-1 high expressed cell lines. With solving of this contravention, we will have a new sight in melanoma and apoptosis.更多还原