露蜂房蛋白［NVP（1）］抑制HepG2细胞增殖的机制研究
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摘要
胡蜂亦称黄蜂,膜翅目(Hymenoptera)胡蜂科(Vespidae)胡蜂属(Polistes),体乌黑发亮,有黄条纹和成对的斑点。胡蜂广泛分布在我国大部分山区,露蜂房为其巢,其中存在大量的抗癌活性物质。研究露蜂房蛋白质对抗癌细胞增殖的药物开发具有重要意义。但露蜂房蛋白质的种类较多,成分复杂,很难分离纯化出一个单一的蛋白质,目前国内外对其研究少有报道,本研究从露蜂房中分离出一个新的蛋白质,并深入地研究了其药理特性,完成的工作主要包括以下几个方面:
1.利用双蒸水长时间4℃浸提,硫酸铵沉淀,0.22μm的滤膜透析脱盐,Sephdex-G50凝胶过滤,反相高效液相色谱层析等方法,从露蜂房中分离纯化出一种蛋白质,质谱分析其分子量为6.6 kD,并定义它为NVP(1)。
2.研究了NVP(1)调节HepG2细胞的增殖机制。6.6μg/ml NVP(1)抑制HepG2细胞增殖作用发生在细胞周期的G1期,并且抑制cyclin B,cyclin D1和cyclin E mRNA的表达,NVP(1)也抑制了cdk2蛋白质的表达,但能增加p27,p21蛋白质的表达;此外,NVP(1)不能明显改变p16蛋白质的表达水平。通过细胞核染色质浓缩检测发现,NVP(1)可促进HepG2细胞凋亡。此外,NVP(1)激活了细胞外调节激酶(ERK)信号通路,当HepG2细胞提前用ERK的特异性抑制剂处理后,p-ERK蛋白质的表达量明显降低。这些结果表明,NVP(1)抑制HepG2细胞增殖是通过激活ERK信号通路,NVP(1)有可能成为肝癌治疗的一种潜在的药物。
3.研究rSK3,rIK通道在细胞增殖中的作用。NVP(1),能抑制Hek-293细胞增殖。Southern-blot检测转基因rSK3,rIK成功,转rSK3,rIK基因后,能消减NVP(1)抗细胞增殖的作用,rSK3,rIK促进细胞增殖。
4.研究了NVP(1)对大鼠支气管平滑肌细胞增殖的抑制作用及其机制。用原代细胞培养,MTT法,流式细胞和免疫印迹检测细胞增殖。发现6.4×10~(-3)g/L的NVP(1)可明显抑制支气管平滑肌细胞增殖,并且阻断在细胞周期的G_1期,对照组G_1期为64.6%,而NVP(1)处理组为90.5%。NVP(1)能增强细胞周期蛋白激酶抑制剂(p21)蛋白质的表达,降低细胞周期蛋白激酶(cdk2)蛋白质的表达。NVP(1)阻断支气管平滑肌细胞增殖于细胞周期的G_1期。
Wasp is a kind of insect with black body,yellow stria and binate speckle of the order Hymenoptera,family Vespidae and genus Polistes.Wasp is widely distributed in most of the mountain of China whose nest,Nidus Vespae contains a lot of anti-cancer component. It is significant to study Nidus Vespae protein in order to find a new nature anti-proliferation drug.However Nidus Vespae protein is difficult to be isolated and purified into single protein.Very few studies at present have been reported on this aspect because of its complicated compositions.A new protein was isolated from Nidus Vespae, which has the activity of inhibiting the proliferation of HepG2 hepatomcells.The mechanisms of its action were studied in the present study and the main results are as follows:
1.A new protein named NVP(1) was isolated from Nidus Vespae after lixiviated in 4℃for 48 h,precipitated by ammonium sulfate,dialyzed by filtration membrane of 0.22μm,separated by Sephdex-G50 gel and reversed-phase HPLC method.
2.The aim of the present study was to elucidate whether and how NVP(1) modulates the proliferation of HepG2 cells.NVP(1) at a concentration of 6.6μg/ml could arrest the cell cycle at stage G_1 and inhibit the mRNA expression of cyclin B,cyclin D1 and cyclin E.NVP(1) suppressed cdk2 protein expression,while increased p27 and p21 protein expression.On the other hand,NVP(1) did not alter p16 protein expression level.NVP(1) promoted apoptosis in HepG2 cells which was indicated by nuclear chromatin condensation.In addition,the ERK kinase signaling pathway was activated.Moreover,the p-ERK protein expression level was attenuated when the HepG2 cells were pretreated with ERK inhibitor PD98059.These results demonstrated that NVP(1) inhibits proliferation of HepG2 through ERK signaling pathway.NVP(1) could be a potential drug for liver cancer.
3.We investigated the effect of rSK3 and rIK anti-NVP(1) in inhibiting proliferation of Hek-293.NVP(1) could inhibit proliferation of Hek-293 cell.At the same time,the effect of rSK3 and rIK anti-NVP(1) on Hek-293 cells proliferation was investigated by cell count assay.Transfection and Southern-blot were used to detect the rSK3 and rIK gene.rSK3 and rIK gene could prompt proliferation and inhibit the effect of NVP(1). rSK3 and rIK channel could prompt proliferation of Hek-293.SK may be a novel target on cancer therapy.
4.We found that NVP(1) could inhibit proliferation of bronchial smooth muscle cell. At the same time,we investigated the mechanism of NVP(1) in inhibiting proliferation of bronchial smooth muscle cell.The effect of NVP(1) on bronchial smooth muscle cells proliferation was assyed by primary cell culture and MTT method.The cell cycle was detected by flow cytometry.Western-blotting was used to examine p21,cdk2 protein expression.NVP(1)(6.4×10~(-3) g/L) could arrest bronchial smooth muscle cell in cell cycle G1 and increase the expression level of p21.On the contrary,NVP(1) could inhibit proliferation of bronchial smooth muscle cell by down-regulating cdk2 protein expression. NVP(1) inhibits proliferation of bronchial smooth muscle cells and induces G1 cell cycle arrest.
1.利用双蒸水长时间4℃浸提,硫酸铵沉淀,0.22μm的滤膜透析脱盐,Sephdex-G50凝胶过滤,反相高效液相色谱层析等方法,从露蜂房中分离纯化出一种蛋白质,质谱分析其分子量为6.6 kD,并定义它为NVP(1)。
2.研究了NVP(1)调节HepG2细胞的增殖机制。6.6μg/ml NVP(1)抑制HepG2细胞增殖作用发生在细胞周期的G1期,并且抑制cyclin B,cyclin D1和cyclin E mRNA的表达,NVP(1)也抑制了cdk2蛋白质的表达,但能增加p27,p21蛋白质的表达;此外,NVP(1)不能明显改变p16蛋白质的表达水平。通过细胞核染色质浓缩检测发现,NVP(1)可促进HepG2细胞凋亡。此外,NVP(1)激活了细胞外调节激酶(ERK)信号通路,当HepG2细胞提前用ERK的特异性抑制剂处理后,p-ERK蛋白质的表达量明显降低。这些结果表明,NVP(1)抑制HepG2细胞增殖是通过激活ERK信号通路,NVP(1)有可能成为肝癌治疗的一种潜在的药物。
3.研究rSK3,rIK通道在细胞增殖中的作用。NVP(1),能抑制Hek-293细胞增殖。Southern-blot检测转基因rSK3,rIK成功,转rSK3,rIK基因后,能消减NVP(1)抗细胞增殖的作用,rSK3,rIK促进细胞增殖。
4.研究了NVP(1)对大鼠支气管平滑肌细胞增殖的抑制作用及其机制。用原代细胞培养,MTT法,流式细胞和免疫印迹检测细胞增殖。发现6.4×10~(-3)g/L的NVP(1)可明显抑制支气管平滑肌细胞增殖,并且阻断在细胞周期的G_1期,对照组G_1期为64.6%,而NVP(1)处理组为90.5%。NVP(1)能增强细胞周期蛋白激酶抑制剂(p21)蛋白质的表达,降低细胞周期蛋白激酶(cdk2)蛋白质的表达。NVP(1)阻断支气管平滑肌细胞增殖于细胞周期的G_1期。
Wasp is a kind of insect with black body,yellow stria and binate speckle of the order Hymenoptera,family Vespidae and genus Polistes.Wasp is widely distributed in most of the mountain of China whose nest,Nidus Vespae contains a lot of anti-cancer component. It is significant to study Nidus Vespae protein in order to find a new nature anti-proliferation drug.However Nidus Vespae protein is difficult to be isolated and purified into single protein.Very few studies at present have been reported on this aspect because of its complicated compositions.A new protein was isolated from Nidus Vespae, which has the activity of inhibiting the proliferation of HepG2 hepatomcells.The mechanisms of its action were studied in the present study and the main results are as follows:
1.A new protein named NVP(1) was isolated from Nidus Vespae after lixiviated in 4℃for 48 h,precipitated by ammonium sulfate,dialyzed by filtration membrane of 0.22μm,separated by Sephdex-G50 gel and reversed-phase HPLC method.
2.The aim of the present study was to elucidate whether and how NVP(1) modulates the proliferation of HepG2 cells.NVP(1) at a concentration of 6.6μg/ml could arrest the cell cycle at stage G_1 and inhibit the mRNA expression of cyclin B,cyclin D1 and cyclin E.NVP(1) suppressed cdk2 protein expression,while increased p27 and p21 protein expression.On the other hand,NVP(1) did not alter p16 protein expression level.NVP(1) promoted apoptosis in HepG2 cells which was indicated by nuclear chromatin condensation.In addition,the ERK kinase signaling pathway was activated.Moreover,the p-ERK protein expression level was attenuated when the HepG2 cells were pretreated with ERK inhibitor PD98059.These results demonstrated that NVP(1) inhibits proliferation of HepG2 through ERK signaling pathway.NVP(1) could be a potential drug for liver cancer.
3.We investigated the effect of rSK3 and rIK anti-NVP(1) in inhibiting proliferation of Hek-293.NVP(1) could inhibit proliferation of Hek-293 cell.At the same time,the effect of rSK3 and rIK anti-NVP(1) on Hek-293 cells proliferation was investigated by cell count assay.Transfection and Southern-blot were used to detect the rSK3 and rIK gene.rSK3 and rIK gene could prompt proliferation and inhibit the effect of NVP(1). rSK3 and rIK channel could prompt proliferation of Hek-293.SK may be a novel target on cancer therapy.
4.We found that NVP(1) could inhibit proliferation of bronchial smooth muscle cell. At the same time,we investigated the mechanism of NVP(1) in inhibiting proliferation of bronchial smooth muscle cell.The effect of NVP(1) on bronchial smooth muscle cells proliferation was assyed by primary cell culture and MTT method.The cell cycle was detected by flow cytometry.Western-blotting was used to examine p21,cdk2 protein expression.NVP(1)(6.4×10~(-3) g/L) could arrest bronchial smooth muscle cell in cell cycle G1 and increase the expression level of p21.On the contrary,NVP(1) could inhibit proliferation of bronchial smooth muscle cell by down-regulating cdk2 protein expression. NVP(1) inhibits proliferation of bronchial smooth muscle cells and induces G1 cell cycle arrest.
引文
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