低氧对LLC-PK_1细胞增殖及去分化的影响

Effect of chronic hypoxia on proliferation and dedifferentiation of LLC PK 1 cells

目的：研究低氧对近端肾小管细胞株（ＬＬＣ－ＰＫ１）细胞增殖和去分化的作用。方法：ＬＬＣ－ＰＫ１细胞分别置于低氧（３％Ｏ２）和正常氧（１８％Ｏ２）孵箱中培养，用３Ｈ－胸腺嘧啶掺入法和细胞计数，观察细胞增殖情况；以钠依赖葡萄糖和氨基异丁酸（ＡＩＢ）转运为指标，观察细胞分化程度；用放射核素法测定蛋白激酶Ｃ（ＰＫＣ）活性。结果：与正常氧培养比较，低氧培养１６ｈ，３Ｈ－胸腺嘧啶掺入显著增加；培养２４ｈ，细胞数显著增加；培养７２ｈ，细胞摄取α－甲基糖甙和ＡＩＢ显著降低。细胞低氧培养４ｈ，细胞膜与细胞质ＰＫＣ活性比率增高，随后降至原先水平；但继续培养至２４ｈ，ＰＫＣ活性再次增高，直至７２ｈ，表明ＰＫＣ呈急性和持续双相激活。ＰＫＣ抑制剂可显著抑制低氧诱导的３Ｈ－胸腺嘧啶掺入，并显著增加α－甲基糖甙和ＡＩＢ转运。结论：慢性低氧可诱导ＬＬＣ－ＰＫ１细胞的增殖和去分化。

Objective: To explore the effect of chronic hypoxia on the proliferation and dedifferentiation of LLC PK 1 cells. Methods: The cells were either exposed to hypoxia(3% O 2) or maintained in normoxia(18% O 2) followed by the assessment of thymidine incorporation and cell number as indices of cellular proliferation and sodium dependent transport of glucose and a minoisobutyric acid(AIB) as indices of differentiation. The activity of protein kinase C(PKC) was determined with radionuclear technique. Results: Exposure of quiescent cultures to hypoxia for 16 h resulted in a significant increase in thymidine followed by a significant increase in cell number at 24 h in comparison with respective normoxic controls. Confluent cultures exposed to 72 h of hypoxia exhibited significant inhibition of α methyl glucose and AIB uptakes when compared with their respective normoxic counterparts. Hypoxia also activated PKC at 4 h followed by a subsequent return to baseline with reactivation at 24 h which remained sustained up to 72 h, suggesting a biphasic acute and sustained activation of PKC. Furthermore, the hypoxia induced alterations in thymidine incorporation as well as α methyl glucose and AIB transport activities were mitigated by inhibitors of PKC. Conclusion: Chronic hypoxia induces both proliferation and dedifferentiation of LLC PK 1 cells mediated, in part, by the sustained activation of PKC.