肝再生增强因子调控细胞增殖及其与钠、钾ATP酶关系研究

发布时间：2018-01-01 16:19

本文关键词：肝再生增强因子调控细胞增殖及其与钠、钾ATP酶关系研究　出处：《华南理工大学》2006年博士论文　论文类型：学位论文

【摘要】：肝再生增强因子(Augmenter of Liver Regeneration ALR)是一种能保护肝脏和特异性促进肝脏细胞增殖的细胞因子，极具潜质成为治疗肝病的基因工程药物。已有的研究表明，ALR与Na~+，K~+-ATPase在体内、体外都能直接结合。但在肝脏细胞中，ALR促进细胞增殖与Na~+，K~+-ATPase的关系未见有相关报道，本文就此问题展开研究，得到以下结果。在细胞水平，采用MTT、[~3H]-TdR掺入和流式细胞仪等方法测定重组人ALR蛋白对细胞增殖的影响。ALR能以浓度依赖效应加速来源于肝脏细胞的DNA合成，改变细胞周期，促进细胞增殖，其起效浓度是50μg／L，最佳的作用浓度范围是100μg／L～200μg／L。抗ALR单克隆抗体能有效阻断ALR的上述作用。对本研究中采用的其他来源的细胞，ALR无促进其增殖的作用。对于HepG2细胞，ALR促进其增殖与Na~+，K~+-ATPase的酶活密切相关，部分抑制Na~+，，K~+-ATPase活性，能够减弱ALR促细胞增殖作用；若完全抑制Na~+，K~+-ATPase活性，ALR对细胞增殖不产生影响。酶活力测定和蛋白免疫印迹证实：ALR能以浓度—时间效应的方式提高细胞Na~+，K~+-ATPase的酶活。半最大刺激浓度为42±11μg／L，ALR对HepG2细胞Na~+，K~+-ATPase的影响表现为短时间大量激活，在5min刺激时，细胞Na~+，K~+-ATPase的酶活达到最大。在ALR的刺激下，HepG2细胞Na~+，K~+-ATPase的V_(max)由0.84±0.11μmol.mg~(-1).min~(-1)上升到1.68±0.07μmol.mg~(-1).min~(-1)(p＜0.01)，而K_m则由23.54±0.12mmol／L减小到20.86±0.13mmol／L(p＞0.05)。激酶抑制剂阻断表明Na~+，K~+-ATPase的磷酸化是ALR提高其转化效率的关键因素，其中以丝氨酸／苏氨酸残基磷酸化为主。抗ALR单克隆抗体能有效阻断ALR提高HepG2细胞Na~+，K~+-ATPase的酶活。荧光探针分析表明：ALR能提高线粒体膜电位，增加细胞内游离[Ca~(2+)]浓度，清除细胞内活性氧。ALR能减缓因Na~+，K~+-ATPase活性下降而造成的对细胞线粒体膜电位的抑制和细胞内游离[Ca~(2+)]浓度的下调，有助于细胞内活性氧的清除。在分子水平上，在50μg／L～200μg／L范围内，ALR能够浓度—时间效应激活MAPK通路，最大激活时间是10min。以浓度效应方式上调细胞周期蛋白CyclinD1表达水平，降低p21表达，提高pRb磷酸化水平。即：ALR通过调控MAPK途径和细胞周期蛋白的作用促进HepG2细胞增殖。用奎巴因抑制Na~+，K~+-ATPase活性，能下调ALR引起的ERK磷酸化水平，上调p38的磷酸化；同时导致Cyclin D1蛋白下调，升高p21表达，pRb蛋白活性下降，表明其抑制了
[Abstract]:The augmenter of Liver Regeneration ALR is a cytokine that can protect the liver and specifically promote the proliferation of liver cells. Studies have shown that ALR and Na ~ + K ~ -ATPase can bind directly in vivo and in vitro, but in liver cells. The relationship between the proliferation of ALR and Na ~ + K ~ -ATPase is not reported in this paper. At the cell level, MTT, [The effect of recombinant human ALR protein on cell proliferation was determined by incorporation of TDR and flow cytometry. ALR accelerated DNA synthesis from liver cells in a dose-dependent manner. The cell cycle was changed and the cell proliferation was promoted. The effective concentration was 50 渭 g / L. The best concentration range is 100 渭 g / L ~ 200 渭 g 路L ~ (-1). Monoclonal antibody against ALR can effectively block the above effects of ALR. ALR had no effect on the proliferation of HepG2 cells. It was closely related to the enzyme activity of Na ~ + K ~ -ATPase, and partly inhibited Na ~ (2 +). K ~ -ATPase activity could attenuate the effect of ALR on cell proliferation. If the activity of Na ~ + -K ~ -ATPase was completely inhibited, ALR had no effect on cell proliferation. Assay of enzyme activity and Western blot showed that the cell Na ~ ~ was enhanced by the concentration-time effect of WALR. The enzyme activity of K ~ -ATPase. The semi-maximal stimulating concentration was 42 卤11 渭 g / L of ALR on the Na ~ + of HepG2 cells. The effect of K ~ -ATPase was a large amount of activation in a short time. After 5 minutes of stimulation, the enzyme activity of Na ~ + -K ~ -ATPase reached the maximum. The activity of Na ~ -ATPase reached the maximum under the stimulation of ALR. HepG2 cells Na ~. The value of K ~ -ATPase increased from 0.84 卤0.11 渭 mol 路mg ~ (-1) to 1.68 卤0.07 渭 mol 路mg ~ (-1). (P < 0.01). The number of km decreased from 23.54 卤0.12 mmol / L to 20.86 卤0.13 mmol / L > 0.05 渭 mol / L, and the blockade of kinase inhibitor indicated that Na ~. Phosphorylation of K ~ -ATPase is the key factor to improve the conversion efficiency of ALR. Among them, serine / threonine residues were mainly phosphorylated, and anti ALR monoclonal antibody could effectively block the enzyme activity of Na ~ + K ~ -ATPase in HepG2 cells enhanced by ALR. Fluorescence probe analysis showed that WALR could increase mitochondrial membrane potential and increase intracellular dissociation. [(Ca~(2)] concentration, scavenging intracellular reactive oxygen species. ALR can slow down the inhibition of mitochondrial membrane potential and intracellular dissociation caused by the decrease of Na ~ + K ~ -ATPase activity. [The down-regulation of the concentration of Ca~(2) is helpful to the removal of reactive oxygen species in cells. At the molecular level, in the range of 50 渭 g / L ~ 200 渭 g / L, ALR can activate the MAPK pathway in a concentration-time effect. The maximum activation time was 10 min. The expression of cyclin CyclinD1 was up-regulated and the expression of p21 was decreased in a concentration-dependent manner. To increase the phosphorylation level of pRb, that is, to promote the proliferation of HepG2 cells by regulating the MAPK pathway and cyclin, and to inhibit the proliferation of HepG2 cells with quinine. K ~ -ATPase activity could down-regulate the phosphorylation level of ERK induced by ALR and up-regulate the phosphorylation of p38. At the same time, Cyclin D1 protein was down-regulated and the activity of p21 protein was decreased, indicating that it inhibited the expression of PRB protein.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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