Cloning of tissue-specific expressing genes in huma ovarian cancer cells and basic study of the adenovirus-mediaetd p16 gene therapy.

卵巢透明细胞腺癌是由于化疗耐药和/或早期淋巴结转移所致的预后较差的组织亚型。它有时受日本人子宫内膜异位症和常见亚型的影响。另一方面，卵巢浆液性腺癌是常见的上皮性癌组织亚型，虽然腹膜转移频繁，但对化疗高度敏感。我们试图通过差异显示的方法寻找透明细胞癌中的特异性表达基因，并将SP17基因确定为候选基因之一。由于SP17基因的功能未知，因此将其全长插入到腺病毒载体中，并感染浆液性腺癌细胞。作为进一步的验证性实验，在透明细胞腺癌中引入了SP17基因的RNAi干扰，并对SP17基因的功能进行了分析。对卵巢CaN…进行MTS检测和细胞死亡检测更多的癌细胞，无论有没有紫杉醇治疗，对卵巢癌最有用的抗癌药物。P16INK4A基因是一种细胞周期蛋白依赖性的激酶抑制因子，是肿瘤抑制基因之一，是细胞周期G1-S进入的关键分子。在约30%的卵巢上皮性癌中，p16基因功能缺失是由于启动子区的杂合性丢失、甲基化和编码区的错义突变所致。将其插入到腺病毒载体中，作为基因辅助治疗的基础实验。为了控制p16基因的功能，我们构建了缺失DNA结合域的ER基因，并将其与p16基因的全长结合。ER-p16融合蛋白只有在雌激素处理条件下才能表达，被认为适合体内应用。从理论上讲，p16功能只有在雌激素存在的条件下才能表达。由于雌激素受体内化携带p16蛋白，p16基因引起的G1期细胞周期停滞仅在雌激素阳性条件下观察到。通过四甲基偶氮唑盐比色法观察到缺乏野生型p16基因的卵巢癌细胞在雌激素存在的条件下，由于感染了ER-p16融合蛋白而发生生长停滞。Western Blot分析证实了雌激素刺激下的细胞内开关系统。报道了基因治疗应用的可能性。较少

Clear cell adenocarcinoma of the ovary is the poor prognostic tissue subtypes due to the chemo-resistance and/or the early lymph node metastases. It is sometimes affected from the endometriosis and frequent subtypes in Japanese. On the other hand, serous adenocarcinoma of the ovary is common epithelial cancer tissue-subtypes, and it is highly sensitive for the chemotherapy though the peritoneal dissemination is frequent. We tried to identify the specifically expressing genes in clear cell adenocarcinoma by using Differential Display method and SP17 gene was identified as one candidate gene. The function of SP17 gene was unknown, so the full length of SP17 gene was inserted into the Adenovirus vector, and it was infected to the serous adenocarcinoma cells. As further confirmative experiment, RNAi interference of SP17 gene was introduced in the clear cell adenocarcinoma, and the function of SP17 gene was analyzed. MTS assay and Cell-death detection assay were performed in the ovarian can … More cer cells, with or without treatment of Paclitaxel, most useful anticancer drug for the ovarian cancers. As results, it was strongly suggested that SP17 plays an important role of chemotherapy-resistance of clear cell adenocarcinoma of the ovary.P16INK4A gene is a cyclin-dependent kinase inhibitor, one of the tumor suppressor genes, and a key molecule of cell cycle G1-S entry. P16 gene function was loss in about 30% of the epithelial ovarian cancers, due to the loss of heterozygosity, methylation of the promoter region and some missense mutations of coding regions. It was inserted into Adenovirus vector as a fundamental experiment of the gene supplementary therapy. In order to control the function of p16 gene, we made the construct of the ER gene which was lack of DNA binding domain combined with full length of p16 gene. The ER-p16 fusion protein which function would be expressed in only estrogen-treatment condition, was considered to suitable for in vivo application. Theoretically, p16 function was expressed only under the estrogen existence condition. The G1 cell cycle arrest due to p16 gene was observed only in estrogen positive condition because of the estrogen receptor internalization carrying p16 protein. The growth arrest of ovarian cancer cells lack of wild type p16 gene was observed by MTT assay only in estrogen existence condition due to infected ER-p16 fusion proteins. Intracellular switching system by the estrogen stimulus was confirmed by Western Blot analyses. The possibility to the gene therapy application was reported. Less