活血散结方药物血浆对PDGF干预下兔视网膜色素上皮细胞增殖相关因子及ERK1/2信号转导通路的影响
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摘要
目的观察活血散结方对血小板源性生长因子(PDGF)干预下兔视网膜色素上皮(RPE)细胞增殖的影响,探讨其分子机制。方法以RPE细胞为研究对象,体外进行RPE细胞原代培养及传代,建立PDGF干预下兔RPE细胞增殖模型,并选取PDGF最佳干预浓度。制备活血散结方药物血浆,对兔RPE细胞增殖与毒性的影响进行测定,选取最佳的实验浓度。实验分为空白对照组(DMEM)、正常血浆组、PDGF(10μg/L)组、PDGF(10μg/L)+AG1296(10μmol/L)组、PDGF(10μg/L)+10%药物血浆组、PDGF(10μg/L)+20%药物血浆组。分别加入相应处理因素干预48 h,Transwell流式细胞仪检测细胞周期变化;Western blot检测兔RPE细胞cyclinD1蛋白、pERK1/2及ERK1/2蛋白表达。结果 10%、20%活血散结方药物血浆对RPE细胞周期、cyclin D1蛋白及p ERK1/2、ERK1/2蛋白有抑制作用,与AG1296抑制剂作用相当(P>0.05),与其他组比较差异均有统计学意义(P<0.01)。结论活血散结方药物血浆可能通过影响ERK信号通路及对周期蛋白的调控,抑制PDGF干预下兔RPE细胞增殖。
Objective To investigate the effect of plasma containing invigorating blood and dissipating masses Chinese medicine on proliferation of rabbit RPE cells treated with PDGF; To discuss the molecular mechanism. Methods The RPE cells were used as the research object and the primitive culture and subculture of RPE cells were proceeded. Rabbit model of RPE cell proliferation treated with PDGF was established, and the best PDGF intervention concentration was chosen. Plasma containing invigorating blood and dissipating masses Chinese medicine was prepared. The effects of rabbit RPE cell proliferation and toxicity were determined, and the best experimental concentration was selected. The experiment was divided into blank control group(DMEM), normal plasma group, PDGF(10 μg/L) group, PDGF(10 μg/L) + AG1296(10 μmol/L) group, PDGF(10 μg/L) + 10% drug plasma group, and PDGF(10 μg/L) + 20% drug plasma group, and corresponding factors were added into each group for intervening for 48 hours. Transwell flow cytometry was used to detect cell cycle changes in different groups. Western blot was used to detect cyclin D1 protein, pERK1/2 and ERK1/2 protein expressions in rabbit RPE cells. Results 10% and 20% concentration of plasma containing invigorating blood and dissipating masses Chinese medicine inhibited RPE cell cycle, cyclin D1 protein, p ERK1/2 and ERK1/2 proteins, which were equivalent to AG1296 inhibitor(P>0.05). There was statistical significance compared with other groups(P<0.01). Conclusion Plasma containing invigorating blood and dissipating masses Chinese medicine may inhibit proliferation of rabbit RPE cells treated with PDGF by affecting ERK signaling pathway and regulation of cyclin.
Objective To investigate the effect of plasma containing invigorating blood and dissipating masses Chinese medicine on proliferation of rabbit RPE cells treated with PDGF; To discuss the molecular mechanism. Methods The RPE cells were used as the research object and the primitive culture and subculture of RPE cells were proceeded. Rabbit model of RPE cell proliferation treated with PDGF was established, and the best PDGF intervention concentration was chosen. Plasma containing invigorating blood and dissipating masses Chinese medicine was prepared. The effects of rabbit RPE cell proliferation and toxicity were determined, and the best experimental concentration was selected. The experiment was divided into blank control group(DMEM), normal plasma group, PDGF(10 μg/L) group, PDGF(10 μg/L) + AG1296(10 μmol/L) group, PDGF(10 μg/L) + 10% drug plasma group, and PDGF(10 μg/L) + 20% drug plasma group, and corresponding factors were added into each group for intervening for 48 hours. Transwell flow cytometry was used to detect cell cycle changes in different groups. Western blot was used to detect cyclin D1 protein, pERK1/2 and ERK1/2 protein expressions in rabbit RPE cells. Results 10% and 20% concentration of plasma containing invigorating blood and dissipating masses Chinese medicine inhibited RPE cell cycle, cyclin D1 protein, p ERK1/2 and ERK1/2 proteins, which were equivalent to AG1296 inhibitor(P>0.05). There was statistical significance compared with other groups(P<0.01). Conclusion Plasma containing invigorating blood and dissipating masses Chinese medicine may inhibit proliferation of rabbit RPE cells treated with PDGF by affecting ERK signaling pathway and regulation of cyclin.
引文
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[12]司艳芳,王君,关娟,等.血小板来源的生长因子通过ERK/p38/PI3K促进人视网膜色素上皮细胞的增殖、迁移[J].现代生物医学进展,2012:12(34):6628-6631.
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[16]GAO J,ZHU J,LI H Y,et a1.Small interfering RNA targeting integrin linked kinase inhibited the growth and induced apoptosis inhuman bladder cancer cells[J].Int J Biochem Cell Biol,2011,43:1294-1304.
[17]PERSAD S,ATTWELL S,GRAY V,et a1.Inhibition of integrinlinked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells[J].Proc Natl Acad Sci USA,2000,97:3207-3212.
[18]ZHU X Y,LIU N,LIU W,et a1.Silencing of the integrin-linked kinase gene suppresses the proliferation,migration and invasion of pancreatic cancer cells(Panc.1)[J].Genet Mol Biol,2012,35:538-544.
[19]D'AMICO M,HULIT J,AMANATULLAH D F,et a1.The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein dependent pathways[J].J Biol Chem,2000,275:32649-32657.
[20]WANG D L,LAN J H,CHEN L,et al.Integrin-linked kinase functions as a tumor promoter in bladder transitional cell carcinoma[J].Asian Pac J Cancer Prev,2012,13:2799-2806.
[21]郭丽莉,于文贞,黎晓新,等.整合素连接激酶对视网膜色素上皮细胞生长、凋亡和分泌功能的影响[J].中华实验眼科杂志,2013,31(8):729-733.
[2]]苗景鹏,魏文斌斌.增生性玻璃体视视网膜病变的发病病机制及治疗研究进展展[J].国际眼科纵纵览,2013,37(6):367-373.
[3]]韩瑞芳.增生性性玻璃体视网膜病病变相关细胞因子子研究进展[J].中华实验验眼科杂志,20166,34(8):765-7688.
[4]]杨晓旭,杨冬萍萍,俞洋.中医药防防治增殖性玻璃体体视网膜病变研究进展展[J].辽宁中医药药大学学报,2017,,19(12):109-1112.
[5]]彭俊,曾志成,谭谭涵宇,等.眼科活活血利水法的基础研究进展[J].眼科新新进展,2010,30((6):585-589,5933.
[6]]胡健艳,陈雪,陈陈辉,等.兔视网膜膜色素上皮细胞培养和鉴定[J].南通大大学学报,2005,225(3):171-173.
[7]]陈吉,彭清华,邢邢雁飞,等.活血利利水法对外伤性增增生性玻璃体视网膜病病变兔眼玻璃体TGF-β1表达的的影响[J].湖南中医药大学学报,2010,30(5):12-15.
[8]陈吉,彭清华,邢雁飞,等.活血利水法对外伤性PVR兔眼玻璃体FNmRNA表达的影响[J].国际眼科杂志,2009,9(12):2303-2306.
[9]付美林,彭清华,陈吉,等.活血利水法对兔外伤性PVR增殖膜上EGFmRNA表达的影响[J].国际眼科杂志,2012,12(1):25-29.
[10]付美林,彭清华,陈吉,等.活血利水法对兔外伤性玻璃体视网膜病变增殖膜上整合素β1表达的影响[J].中华中医药学刊,2011,29(4):722-725.
[11]PUNN A,MOCRIDGE J W,FAROOQUI S,et al.Sustained activation of p42/p44 mitogen-activated protein kinase during recovery from simulated ischaemia mediates adaptive cytoprotection in cardiomyocytes[J].Biochem J,2000,350:891-899.
[12]司艳芳,王君,关娟,等.血小板来源的生长因子通过ERK/p38/PI3K促进人视网膜色素上皮细胞的增殖、迁移[J].现代生物医学进展,2012:12(34):6628-6631.
[13]MOTKURA T,BLOOM T,KIM H G,et al.A novel cyclinencoded by a bell-linked candidate oncogene[J].Nature,1991,350:512.
[14]MARX J.How cells cycle toward cancer[J].Science,1994,263:319-321.
[15]SHERR C J.G1 phase progression:cycling on cue[J].Cell,1994,79:551-555.
[16]GAO J,ZHU J,LI H Y,et a1.Small interfering RNA targeting integrin linked kinase inhibited the growth and induced apoptosis inhuman bladder cancer cells[J].Int J Biochem Cell Biol,2011,43:1294-1304.
[17]PERSAD S,ATTWELL S,GRAY V,et a1.Inhibition of integrinlinked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells[J].Proc Natl Acad Sci USA,2000,97:3207-3212.
[18]ZHU X Y,LIU N,LIU W,et a1.Silencing of the integrin-linked kinase gene suppresses the proliferation,migration and invasion of pancreatic cancer cells(Panc.1)[J].Genet Mol Biol,2012,35:538-544.
[19]D'AMICO M,HULIT J,AMANATULLAH D F,et a1.The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein dependent pathways[J].J Biol Chem,2000,275:32649-32657.
[20]WANG D L,LAN J H,CHEN L,et al.Integrin-linked kinase functions as a tumor promoter in bladder transitional cell carcinoma[J].Asian Pac J Cancer Prev,2012,13:2799-2806.
[21]郭丽莉,于文贞,黎晓新,等.整合素连接激酶对视网膜色素上皮细胞生长、凋亡和分泌功能的影响[J].中华实验眼科杂志,2013,31(8):729-733.