温熱刺激によるグリオーマ細胞回転の変化と熱ショック蛋白（70kDa）の発現

Alterations in Cytokinetics and Heat Shock Protein (70kDa) Expression of Glial Cell by Hyperthermia

　1）グリア系腫瘍細胞としてラットグリオーマC6，354A，ヒトグリオブラストーマT98G，A172，正常細胞としてラットグリア芽細胞を用い，温熱刺激による細胞動態の変化および熱ショック蛋白質（HSP70）発現についてフローサイトメトリーおよびウエスタンブロッテング法を用いて調べた。

　2）温熱刺激によりグリオーマではS期またはG₂／M期に集積する細胞回転が示されたが，グリア芽細胞では著明な変化はなかった。HSP70発現はいずれの細胞株とも温度および時間依存性に漸増が認められ，さらに温熱刺激による細胞回転（特にS期）への影響が強い細胞ほど温度上昇によるHSP発現の増加率が大きくなる可能性がある。

　3）フローサイトメトリーによる二色解析法を用いたC6細胞の細胞周期とHSP70発現との解析ではG₂／M，S，G₀／G₁期の順に陽性率が高く，HSPはG2／M期に最も強く発現し，細胞周期により発現が異なる可能性が示唆された。

Expression of major heat shock and stress-in-duced protein, HSP70, is known to be under com-plex regulation in tumor cells. In this study, we investigated the alternations of cytokinetics and HSP70 expression by hyperthermia in the invitro experimental systems, using two rat glioma cell lines, two human glioblastoma cell lines and rat glioblast cells. For hyperthermal treatment the flasks were placed in water baths warmed up at 41 -45℃ for 15 min. To determine the effect of hyper-thermia on the cell cycle progression, the changes in the DNA distribution of the cell population werestudied by flow cytometry (FCM). The levels of HSP70 protein were determined by immunoblot analysis. The relationship between cell cycle and HSP70 expression was investigated by FCM using PI and FITC-labelled HSP70 double staining tech-nique. These results were as follows : 1) Compared with the control, hyperthermic treatment at 42℃ or 44℃ caused both 354A and T98G cells to accumu-late in S phase 18 hours after treatment and G₂/M phase after 6-18 hours. 2) Hyperthermic treatment at 42℃ caused C6 cells to accumulate in S phase 6 hours after treatment, whereas heat treatment at 44℃ caused C6 cells to accumulate in S phase after 18 hours and G₂/M phase after 6 hours. 3) A172 cells were accumulated only in G₂/M phase by hyperthermia. 4) Glioblast cells did not show the alterations of cytokinetics by heat treatment remarkably. 5) HSP70 protein synthesis were en-hanced under hyperthermic conditions in all type of cells, whether primary glioblast or permanent glioma cell lines. The increase of HSP protein synthesis was particularly enhanced in the cell lines, which cytokinetics remarkably altered due to S phase block and G₂/M block by heat treatment. 6) The maximum expression of HSP70 was detected in the cells at G₂/M phase, indicating that HSP70 expression would be regulated during the cell cycle.