- 期刊
豬主動脈平滑肌細胞之分離、鑑別與熱休克蛋白質合成
Isolation, Characterization and Heat Shock Protein Synthesis of Porcine Aortic Smooth Muscle Cells
摘要
本研究之目的在分離及確認豬主動脈平滑肌細胞並研究其在加熱處理後熱休克蛋白質合成等特性。由6 個月齡之成豬主動脈所分離得到之平滑肌細胞經由繼代主培養不同代數所得到的生長曲線顯示彼此之間沒有差異。第5至第12次的繼代培養細胞之雙倍時間約為90小時。免疫螢光染色法分析的結果顯示，豬主動脈平滑肌細胞有典型的肌動蛋白和vimentin 的絲狀染色形態。進一步西方免疫吸漬法定量結果顯示在不同培養代數之平滑肌細胞α-smooth muscle actin的量無明顯差異。所得的細胞株經45℃ 60分鐘加熱處理後使其恢復0到12小時，然後以放射性同位素標定法和西方免疫吸漬法來分析細胞熱休克蛋白質合成的質與量。結果清楚指出，熱休克蛋白質70與72可被熱處理所誘導而合成出來，而且在恢復10小時之後可累積至最大量。以上結果指出我們所分離而且經誘導數代的豬主動脈平滑肌細胞具有相當的純度以及相當穩定的生長特性及熱休克反應性，是一個研究熱休克蛋白質與其在疾病形成過程中所扮演角色的很好的體外模式。同時，由於豬與人類心臟血管結構相類似，豬主動脈平滑肌細胞株的成功建立將有助於人類心臟血管疾病之預防與治療之研究。
並列摘要
The purpose of this study was to isolate and identify smooth muscle cells from adult porcine aorta and to characterize this cell strain by studying protein synthesis after heat treatments. There was no significant difference in trend of growth curves between porcine aortic smooth muscle cells (aSMC) subculturcd for various passage numbers. The doubling time of the cells from 5-12 passages was approximate 90 hr. Immunofluorescence staining using α-smooth muscle actin (α-SMA) and vimentin monoclonal antibodies confirmed the morphology of actin filaments and vimentin-containing intermediate filaments in aSMC. Western blot analysis showed no significant difference in the expression level of α-SMA among the different passages. Further studies of protein synthesis in porcine aSMC were followed by 45°C for 60 min heat treatment. After treatments, cells were recovered for up to 12hr and then metabolically labeled with [35S] methionine for 1hr. De novo protein synthesis was monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The subsequent quantitation of 70 kD heat shock protein (HSP70) was performed by Western blotting using antibody against HSP70. The results showed that the synthesis of HSP70 could be heat-induced and was peaked at the period of 4 to 6hr after recovery. Moreover, HSP70 was accumulated to a maximal level after 10 hr recovery. Thus, the porcine aSMC cultures we established had characteristics of high purity, stability, and responsiveness to heat shock response. This in vitro model may provide a valuable tool for the study of heat shock response played in atherosclerosis and restenosis after balloon angioplasty.