ATP对人不死化成纤维细胞DNA的合成、膜蛋白表达及离子通道的影响

Effect of ATP on the DNA synthesis, membrane protein expression and channel inimmortalized human fibroblasts

目的：探讨ATP抑制不死化人成纤维细胞增殖过程中对其DNA合成、细胞膜蛋白的表达以及离子通道的影响。方法：将正常人TIG-7和OUMS-36细胞株，不死化人KMST-6和SUSM-1细胞株在不同浓度的ATP、ADP、AMP，条件下分别进行24和96 h的常规细胞培养，观察存活细胞数目、DNA的合成、［32P］ATP标记膜蛋白的表达以及不死化KMST-6细胞分别在无钙离子培养基和应用Ca2+和K+通道桔抗剂时，细胞增殖率的改变。结果：培养96h后，0．4mmol/L ATP对不死化细胞KMST-6的抑制率为77％，且 DNA合成明显地被抑制，而正常 OUMS-36细胞抑制率为41％，且 DNA合成无明显改变。在 1mmol/L ATP时，多数KMST-6细胞发生死亡，而正常OUMS-36细胞的增殖无明显影响。当ADP、AMP，和腺苷或磷酸处理的细胞，仅有ADP处理的不死化细胞存活数目减少（P<0．01）。与正常细胞比较，不死化细胞30，31，33和40 kD的[32P]标记膜蛋白呈高表达。0．4 mmol/L ATP与 KMST-6细胞共培养时分别加入 Ca2+以及K+通道拮抗剂，这些药物在某种程度上可以降低 ATP的增殖抑制作用，当ATP处理的细胞液中无钙离子时，它们的增殖不受影响。结论：ATP对不死化人成纤维细胞的DNA合成有明显的抑制作用；并且使磷酸化细胞膜蛋白表达增高；其过程中有钙通道和钾通道的参与?

Abstract Objective:To investigate the effect of ATP on proliferation and membrane protein expression of immortalized human fibro-blasts.Methods: Applying the normal human fibroblast cell line TIG-7 and OUMS-36, the immortalized human fibroblast cell line KMST-6 andSUSM-1 to culture 24 h and 96 h with the different concentration of ATP, ADP and AMP respectively, observed the number of cells alive, DNAsynthesis and the expression of [32 P]-ATP labeled proteins in the membrane fraction. Results: After 96 h cultured, the proliferation inhibition ofimmortalized cells exposed to 0.4 mmol/L ATP was 77% compared with that of the control cultures,while that of normal OUMS-36 cells treatedwith the same concentration was 41%;the DNA sythesis of the immortalized cells exposed to 0.4 mmol/L ATP was greater suppressed thanthat of normal OUMS-36 cells. Treated the immortalized cells with ADP, AMP, adenosine, and phosphoric acid, ADP could reducee the number ofcells significantly(P<0.01) In the pressence of 1 mmol/L are, many KMST-6 cells died,but normal OUMS-36 cells did not Compared withnormal cells,30,31,33 and 40 kD proteins were markedly labeled in the immortalized cells. KMST-6 cell were treated with 0.4 mmol/L ATP,ATP combined with calcium or potassium channel antagonists, respectively.These drugs can decrease ATP inhibited the proliferation. When ATPdeal with the culture solution which have no calcium, ther proliferation uninfluenced. Conclusion: ATP inhibited the DNA synthesuis and in-creased membrane protein expression in immortalized human fibroblasts. The calcium and potassium involved in the inhibitory effects of ATP, onthe proliferation signaling in immortalize human fibroblasts.