本文關(guān)鍵詞：肝再生增強(qiáng)因子調(diào)控細(xì)胞增殖及其與鈉、鉀ATP酶關(guān)系研究　出處：《華南理工大學(xué)》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝再生增強(qiáng)因子(ALR) Na~+ K~+-ATP酶 增殖 MAPK 細(xì)胞周期 調(diào)控

【摘要】：肝再生增強(qiáng)因子(Augmenter of Liver Regeneration ALR)是一種能保護(hù)肝臟和特異性促進(jìn)肝臟細(xì)胞增殖的細(xì)胞因子，極具潛質(zhì)成為治療肝病的基因工程藥物。已有的研究表明，ALR與Na~+，K~+-ATPase在體內(nèi)、體外都能直接結(jié)合。但在肝臟細(xì)胞中，ALR促進(jìn)細(xì)胞增殖與Na~+，K~+-ATPase的關(guān)系未見(jiàn)有相關(guān)報(bào)道，本文就此問(wèn)題展開(kāi)研究，得到以下結(jié)果。 在細(xì)胞水平，采用MTT、[~3H]-TdR摻入和流式細(xì)胞儀等方法測(cè)定重組人ALR蛋白對(duì)細(xì)胞增殖的影響。ALR能以濃度依賴效應(yīng)加速來(lái)源于肝臟細(xì)胞的DNA合成，改變細(xì)胞周期，促進(jìn)細(xì)胞增殖，其起效濃度是50μg／L，最佳的作用濃度范圍是100μg／L～200μg／L�？笰LR單克隆抗體能有效阻斷ALR的上述作用。對(duì)本研究中采用的其他來(lái)源的細(xì)胞，ALR無(wú)促進(jìn)其增殖的作用。對(duì)于HepG2細(xì)胞，ALR促進(jìn)其增殖與Na~+，K~+-ATPase的酶活密切相關(guān)，部分抑制Na~+，，K~+-ATPase活性，能夠減弱ALR促細(xì)胞增殖作用；若完全抑制Na~+，K~+-ATPase活性，ALR對(duì)細(xì)胞增殖不產(chǎn)生影響。 酶活力測(cè)定和蛋白免疫印跡證實(shí)：ALR能以濃度—時(shí)間效應(yīng)的方式提高細(xì)胞Na~+，K~+-ATPase的酶活。半最大刺激濃度為42±11μg／L，ALR對(duì)HepG2細(xì)胞Na~+，K~+-ATPase的影響表現(xiàn)為短時(shí)間大量激活，在5min刺激時(shí)，細(xì)胞Na~+，K~+-ATPase的酶活達(dá)到最大。在ALR的刺激下，HepG2細(xì)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則由23.54±0.12mmol／L減小到20.86±0.13mmol／L(p＞0.05)。激酶抑制劑阻斷表明Na~+，K~+-ATPase的磷酸化是ALR提高其轉(zhuǎn)化效率的關(guān)鍵因素，其中以絲氨酸／蘇氨酸殘基磷酸化為主�？笰LR單克隆抗體能有效阻斷ALR提高HepG2細(xì)胞Na~+，K~+-ATPase的酶活。 熒光探針?lè)治霰砻鳎篈LR能提高線粒體膜電位，增加細(xì)胞內(nèi)游離[Ca~(2+)]濃度，清除細(xì)胞內(nèi)活性氧。ALR能減緩因Na~+，K~+-ATPase活性下降而造成的對(duì)細(xì)胞線粒體膜電位的抑制和細(xì)胞內(nèi)游離[Ca~(2+)]濃度的下調(diào)，有助于細(xì)胞內(nèi)活性氧的清除。 在分子水平上，在50μg／L～200μg／L范圍內(nèi)，ALR能夠濃度—時(shí)間效應(yīng)激活MAPK通路，最大激活時(shí)間是10min。以濃度效應(yīng)方式上調(diào)細(xì)胞周期蛋白CyclinD1表達(dá)水平，降低p21表達(dá)，提高pRb磷酸化水平。即：ALR通過(guò)調(diào)控MAPK途徑和細(xì)胞周期蛋白的作用促進(jìn)HepG2細(xì)胞增殖。用奎巴因抑制Na~+，K~+-ATPase活性，能下調(diào)ALR引起的ERK磷酸化水平，上調(diào)p38的磷酸化；同時(shí)導(dǎo)致Cyclin D1蛋白下調(diào)，升高p21表達(dá)，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 鹵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 鹵0.11 渭 mol 路mg ~ (-1) to 1.68 鹵0.07 渭 mol 路mg ~ (-1). (P < 0.01). The number of km decreased from 23.54 鹵0.12 mmol / L to 20.86 鹵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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R341

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