肝再生增强因子对HepG2细胞Na+，K+-ATP酶的影响

华南理工大学学报（自然科学版） ›› 2006, Vol. 34 ›› Issue (5): 58-62.

肝再生增强因子对HepG2细胞Na⁺，K⁺-ATP酶的影响

林莹¹ 佟明华² 孔祥平^2† 梁世中¹

1．华南理工大学生物科学与工程学院，广东广州 510640；2．空军广州医院肝病研究所，广东广州 510600)

收稿日期:2005-07-07 出版日期:2006-05-25 发布日期:2006-05-25
通信作者: 孔祥平，主任医师，E-mail：xiangpmg_kong@hntmail.com E-mail:ly9a9@126.com
作者简介:林莹(1971-)，女，博上牛，主要从事微生物发酵和生物制药研究．
基金资助:
广东省重点科技项目基金(99B04704G-47-1)

Efect of Augmenter of Liver Regeneration on Na⁺，K⁺-ATPase Activity of HepG2 Cell

Lin Ying¹ Tong Ming-hua² Kong Xiang-ping² Liang Shi-zhong¹

1.College of Biological Science and Engineering,South China Univ.of Tech.,Guangzhou 5 10640,Guangdong,China;2.Liver Diseases Research Institute.Guangzhou Hospital of Air-Force,Guangzhou 510602,Guangdong,China

Received:2005-07-07 Online:2006-05-25 Published:2006-05-25
Contact: 孔祥平，主任医师，E-mail：xiangpmg_kong@hntmail.com E-mail:ly9a9@126.com
About author:林莹(1971-)，女，博上牛，主要从事微生物发酵和生物制药研究．
Supported by:
广东省重点科技项目基金(99B04704G-47-1)

摘要/Abstract

摘要： 用四甲基偶氮唑盐比色法(Mr丌法)检验重组肝再生增强因子(ALR)对人肝母细胞瘤细胞系(HepG2)细胞的增殖作用，无机磷比色法测定细胞Na⁺，K⁺-ATP酶的酶活，蛋白质免疫印迹分析Na⁺，K⁺-ATP酶的磷酸化情况．体外实验结果表明，重组ALR能促进HepG2细胞增殖．ALR可提高HepG2细胞Na⁺，K⁺-ATP酶酶活，符合Michaelis-Menten方程作用机制，ALR提高了细胞Na⁺，K⁺-ATP酶的转化效率，V_max由(0．84±0．1 1)μmol/(mg·min)上升到(1．68±0．07)μmol/(mg·min)(P<0．01，差异极显著)，而K 则由(23．54±0．12)mmol/L变为(20．86±0．13)mmol/I (P>0．05，差异无显著性)．丝氨酸/苏氨酸残基磷酸化是酶活提高的关键因素．蛋白质免疫印迹分析也证实：ALR对Na⁺，K⁺-ATP酶磷酸化的影响属于剂量一时间依赖型．

关键词: 肝再生, 增强因子, Na⁺, K⁺-ATP酶, 转化效率, 剂量一时间依赖

Abstract:

The effect of recombinant augmenter of liver regeneration(ALR)on the proliferation of HepG2 cell was investigated by the MTY(Thiazolyl blue)assay,and the activity of the Na ,K -ATPase was measured by an in-organic phosphorus spectrophotometer.Then,the phosphorylation of Na⁺,K⁺-ATPase was discussed by means of protein immunoblot.The results show that recombinant ALR promotes the proliferation of HepG2 cell in vitro.As for the mechanism.the stimulation of Na⁺,K⁺-ATPase in HepG2 cell caused by ALR accords with the Michaelis-Menten equation model.Moreover,ALR improves the turnover rate of Na⁺,K⁺-ATPase-the value of V_max increases from(0.84±0.11)μmol/(mg·min)to(1.68±0.07)μmol/(mg·min)(P<0.01)and that of K_m decreases from(23.54±0.12)mmol/L to(20.86±0.13)mmol/L(P>0.05).Also,the resuhs demonstrate that the phosphorylation of serine or threonine is key to the stimulation of Na⁺,K⁺-ATPase.A dose-time dependence of the phosphorylation of Na⁺,K⁺-ATPase in HepG2 cell stimulated by ALR is finally revealed by means of protein im-munoblot.

Key words: liver regeneration, augmenter, Na⁺,K⁺-ATPase , turnover rate, dose—time dependence

林莹佟明华孔祥平梁世中. 肝再生增强因子对HepG2细胞Na⁺，K⁺-ATP酶的影响[J]. 华南理工大学学报（自然科学版）, 2006, 34(5): 58-62.

Lin Ying Tong Ming-hua Kong Xiang-ping Liang Shi-zhong. Efect of Augmenter of Liver Regeneration on Na⁺，K⁺-ATPase Activity of HepG2 Cell[J]. Journal of South China University of Technology (Natural Science Edition), 2006, 34(5): 58-62.