Basic study for the improvement of melanoma radiotherapy by suppression of TRP-2 gene expression

TRP-2基因在黑色素瘤细胞中特异性表达，被认为与黑色素瘤的放射抗性有关。本项目旨在建立通过抑制TRP-2基因表达改善黑色素瘤放疗的方法，首先进行培养黑色素瘤细胞水平实验。本课题的研究成果总结如下：1。将TRP-2 sh-RNA表达质粒转染酪氨酸酶表达缺陷型黑色素瘤细胞，构建TRP-2表达缺陷型黑色素瘤细胞系。建立了x射线、紫外线和顺铂对黑色素瘤细胞中TRP-2表达影响的杀伤效果评价体系。建立了黑色素单体DHICA含量测定的评价体系，DHICA是TRP-2的产物。相反，为了评估细胞中TRP-2表达的影响，非黑色素瘤培养细胞，用TRP-2表达质粒转染HeLa细胞。利用该细胞系，我们可以明确黑色素瘤的抗辐射能力是否与TRP-2蛋白的表达和产生有关，还是与TRP-2相关的黑色素形成有关。我们评估了这些细胞系在黑色素形成、细胞生物学和分子生物学方面的特性。比较已建立细胞系的这些特性，我们无法得到TRP-2基因抑制提高x射线辐射、紫外线和顺铂杀伤效果的明确结果。目前，我们正在完善评价体系和方法，并继续对效果进行评价。

TRP-2 gene is specifically expressed in melanoma cells, and is thought to related to the radio-resistance of melanoma. The aim of this project is to establish the method of improvement of radiotherapy for melanoma by suppression of TRP-2 gene expression, cultured melanoma cell level experiments were performed as the first step.The results obtained through this research project are summarized as follows :1. TRP-2 sh-RNA expression plasmids were transfected to tyrosinase-ecpression deficient amelanotic melanoma cell to construct TRP-2 expression deficient melanoma cell lines.2. Evaluation systems for the killing effect of X-ray, UV, and cisplatin were established to the effect of TRP-2 expression in melanoma cells.3. Evaluation system for measuring the content of melanin monomer DHICA, which is the product of TRP-2, was established.4. Inversely, to evaluate the effect of TRP-2 expression in cells, non-melanoma cultured cell, HeLa cells were transfected with TRP-2 expression plasmid. Using this cell line we can clarify whether the radiation-resistance of melanoma is related to the expression and production of TRP-2 protein or melanogenesis related to TRP-2. We evaluated the properties of these cell lines concerning melanogenesis, cell biology, and molecular biology.Comparing the these properties of established cell lines, we could not obtained clear results that TRP-2 gene suppression improve the killing effect of X-ray radiation, UV, and cisplatin. Currently, we improve the evaluation systems and methods, and continue to the evaluation of the effects.