Cytofluorometryを用いたグリオーマ細胞の研究—ホルマリン固定パラフィン包埋材料よりDAPI-HP二重蛍光染色法を用いた核内DNA量の測定について

DAPI-DNA CYTOFLUOROMETRIC STUDY OF GLIOMA CELLS : APPLICATION OF DAPI-DNA CYTOFLUOROMETRY TO PARAFFIN EMBEDDED ARCHIVAL GLIOMA TISSUE FOR NUCLEAR DNA CONTENT ANALYSIS

　過去にホルマリン固定パラフィン包埋が行なわれた14例のglioma材料（low grade astrocytoma6，malignant astrocytoma 8）を対象として，DAPI-HP同時二重染色を行ないDAPI-DNA複合体の蛍光強度を測定する事により，組織学的に腫瘍細胞を確認しながら核内DNAを定量することができた。よって，上記gliomaの腫瘍細胞の核DNAを測定し，その核DNA量によりN群（2C相当のDNA量をもつ細胞群，normoploidy），S群（N群未満のDNA量をもつ細胞群，hypoploidy），L群（4C以上の核DNAをもつ細胞群hypertetraploidy），I群（N群とL群の中間の核DNA量をもつ細胞群inter—mediate poidy）の4群に分け，各群の細胞数の全細胞数に対する百分率を算出した。その結果，astrocy—toma grade I，IIと比較すると，glioblastomaではI群intermediate ploidyは有意に高値を，N群normoploidyは有意に低値を示し，組織学的悪性度とDNA蛍光強度との相関を示唆する測定値を得た。しかしL群hypertetraploidyは予想に反して，glioblastomaの方がastorocytoma gradeI，IIより低値であった。この理由については，本測定法の特殊性と密接な関連があり，この点について論議した。本法は過去の手術例のパラフィン包埋材料から，Flow cytometerがなくても，glioma細胞核のDNA量の測定を可能にしたこと，そして過去の手術例の臨床経過のretrospective studyに利用できる可能性が示唆されたことなどに，臨床的な意義があると考えられた。

Using DAPI-DNA cytofluorometry, the author analyzed nuclear DNA content of formalin fixed, paraffin embedded, glioma material obtained from 14 glioma cases at surgery. Sections of 10 Atm were deparaffinized. Following simultaneous DAPI (4, 6-diamidino-2-phenylindole dihydroporphilin chlo-ride)/HP (hematoporphyrin) staining, DAPI binds DNA and DNA-DAPI complexes emit blue fluo-rescence when exited by ultraviolet (UV) light. Through Zeiss fluorescence microscope, the auhtor measured nuclear fluorescence intensity with his-tological verification of glioma cells. A DNAhistogram was obtained with fluorescence inten-sity recorded on the abscissa and number of cells plotted on the ordinate. Samples of 20 normal non-neoplastic astrocytes taken from apparently normal brain tissue included in the histological slide were used as diploid (2C) control. Based on DNA content, tumor cells were classified into 4 groups : N-group composed of cells with 2C DNA content (normoploid), S-group with less than 2C (hypopliod), L-group more than 4C (hypertet-raploid), I-group between 2C and 4C (interme-diate ploid). Intermediate pliod was significantly higher and normoploid was significantly lower in glioblastoma compared with those of benign ast-rocytoma. Thus, DNA content and histologicalmalignancy were well correlated. Due to limita-tion of measuring diaphragm of turret in the mic-roscope, some extralarge cell could not be inc-luded in it and was excluded from the measure-ment. On this account, population of hypertetra-ploid cell in glioblastoma was smaller than that of benign astrocytoma. However, the advantage of this preparation method lies in ( 1 ) nuclear DNA measurement of individual glioma cells with histological identifications and ( 2 ) retrospective studies of glioma patients operated long time ago with regard to clinical behavior and DNA content of the tumor cell nuclei, even when flow cyto-meter is not available.