|Budget Amount *help
¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1995 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Endometrial carcinoma tissues showed locally high activity of estrogen based on high activity of estrtone-sulfatase. However, histrological localization of the enzyme has not been unknown, because anti-cstrone-sulfatase monoclonal antibody is not available yet. The purpose of present study is the production of the monoclonal antibody and in order to demonstrate the High-activity of estrone-sulfatase in endometrial carcinoma tissues has been reported.histological localization of estrone sulfatase in endomentrial tidsses. Ten placentas were homogenized and cluted by columchromatography. Enzyme assay with H3-estrone-sulfate showed a high rich peak of estrone-sulfatase. The protein on the peak were eluted and was used as an antigen for the production of monoclonal antibody. BALB/c nu/nu mice were immunized with the protein and then twelve clone cells were selected by both ELISA and western-blotting. Immunohistochemistry showed that the all monoclonal antibodies were reactive to normal plac
enta, but inactive to placentas of congenital ichtyosis. Transfection with aryl-sufatase-C gene to breast carcinoma cell lines (MCF-7 and T47D) showed higher activities of both estrone-sulfatse and dehydroepiandrosterone than transfectants with vector alone. The monoclonal antibodies reacted on the enzyme proteins from the transfectants. This proves that the monoclonal antibodies were detected both estrone-sulfatase and dehydroepiandrosterone, Immunohistochemical staining of endometrial tissues and neoplasms demonstrated that normal endometrial tissues were inactive, but endometrial acrcinoma tissues, especially carcinoma cells alone, were reactive to the antibody. This suggests that estrone-sulfatase are localized on epithelial cells of endometrial carcinomas. In conclusion, we succeeded in the production of anti-estrone-sulfatase monoclonal antibodies.
正常胎盤10コからカラムクロマトグラフィーでモノピークを示し, かつES活性の高い蛋白分角を抽出した。BALB/Cマウスに抽出蛋白を計3回免疫し,hybridoma cellを作成。cloning後,ELISAとWestern blotでスクリーニングを行い12種の抗体を得た。正常者と先天性魚鮮癬患者の胎盤とで比較免疫組織化学染色を行った。なお胎盤使用には,患者の同意を得た。Aryl sulfatase C遺伝子を乳癌細胞(MCF-7,T470細胞株)に移入し,ESの酵素活性をみたところ,空VECTORのみの遺伝子移入細胞に比し,ESとDehydro epiandro sterone sulfatase(DEAS)の両活性が高値を示した。これら細胞から抽出した蛋白と,MAbとは反応し,胎盤抽出抗原と反応が一致した。この結果は,作製したモノクローナル抗体がESとDEASの両酵素に反応するものと考えられた。正常子宮内膜と内膜癌組織においては,抗ESMAbは,上皮細胞のみに反応し,G_1,G_2,G_3,全ての内膜腺癌組織において腺癌細胞のみが反応し,間質には反応しなかった。