大鼠pEGFP-N1-BKβ_1真核表达载体的构建及苦杏仁苷对支气管平滑肌细胞增殖的研究
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摘要
本论文中通过反复实验得到利用酶消化法稳定获得支气管平滑肌细胞的方法。分离、剪碎的支气管平滑肌,用0.05%Trypsin37℃恒温消化90min左右,辅以吹打,终止消化得到大量带状或纺锤状、细胞膜光滑、细胞质均匀的平滑肌细胞。大约一星期铺满培养瓶底,用含10%FBS的DMEM-F12培养基进行传代培养。
钙激活钾通道(KCa)有大电导的钙激活钾通道(BK)、中电导(MK)和小电导(SK)的钙激活钾通道三种类型,BK通道的β亚基有β_1、β_2、β_3、β_4四种异形体。本论文应用RT-PCR技术,从原代培养的Wistar大鼠支气管平滑肌细胞的第3-5代提取总RNA,逆转录,针对BKβ_1的特异性片断PCR扩增鉴定,发现β_1亚基有表达,然后通过胶回收,连接,转化等步骤将其和pEGFP-N1真核表达载体相连,构建了含有绿色荧光蛋白的pEGFP- N1-BKβ_1融合基因表达载体,准备转染HEK-293细胞,进一步研究β_1亚基的功能。
苦杏仁苷是传统中药苦杏仁中的有效成分,人们对它的研究已有近200年的历史,现已成为医药上常用的祛痰止咳剂、辅助性抗癌药物。在治疗哮喘的麻杏石苷汤,麻黄汤等中药中都有苦杏仁苷的成分。我们利用MTT实验方法也证实了苦杏仁苷对支气管平滑肌细胞的增殖具有微弱的抑制作用。研究表明,以不同浓度的苦杏仁苷作用于BSMC后,在24hrs后开始出现抑制,并且随着浓度的增大抑制作用增强,加药浓度为0.704 mmol/L时抑制率达到50%。
The methods of obtaining bronchus smooth muscle cell (BSMC) were dicussed: digest method.after getting the cells from the wista rat,then 0.05% Trypsin for 1hr and 30mins in 37℃.one week later,cells were full of the base of the bottle.the BSMC were subcultured in DMEM-F12 containing 10% FBS with zobary and spindly smooth membrane.
The Objection of the thesis is to construct green fluorescent protein (pEGFP-N1)- BKβ_1 fusion gene vector for observing the expression and localization of BKβ_1 in HEK-293 cells. the coding region in cDNA was amplified by RT-PCR from rat airway smooth muscle cells and rat bronchia smooth muscle cells.Total RNA is extracted from the 3rd - 5th generation cells,reversely translated,then followed with PCR and recombined into pEGFP-N1 plasmid expressing pEGFP-N1.After identification with restriction endonucleases and sequence analysis,the recombinant plasmid will be transfected into HEK-293 cells with the cationic liposome DOTAP as the transfection reagent.
Amygdalin (D-mandelonitrile-β-D-gentiobioside) is a natural compound with the anti-tussive and anticancer activities.It is decomposed by the action ofβ-D-glucosidase to yield hydrocyanic acid which stimulates the respiratory center reflexively and produces a kind of antitussive and antiasthmatic effects.amygdalin is important in respiratory disease and cardiovascular disease.In this thesis,Amygdalin inhibited BSMC proliferation by 50% after 24hrs.
钙激活钾通道(KCa)有大电导的钙激活钾通道(BK)、中电导(MK)和小电导(SK)的钙激活钾通道三种类型,BK通道的β亚基有β_1、β_2、β_3、β_4四种异形体。本论文应用RT-PCR技术,从原代培养的Wistar大鼠支气管平滑肌细胞的第3-5代提取总RNA,逆转录,针对BKβ_1的特异性片断PCR扩增鉴定,发现β_1亚基有表达,然后通过胶回收,连接,转化等步骤将其和pEGFP-N1真核表达载体相连,构建了含有绿色荧光蛋白的pEGFP- N1-BKβ_1融合基因表达载体,准备转染HEK-293细胞,进一步研究β_1亚基的功能。
苦杏仁苷是传统中药苦杏仁中的有效成分,人们对它的研究已有近200年的历史,现已成为医药上常用的祛痰止咳剂、辅助性抗癌药物。在治疗哮喘的麻杏石苷汤,麻黄汤等中药中都有苦杏仁苷的成分。我们利用MTT实验方法也证实了苦杏仁苷对支气管平滑肌细胞的增殖具有微弱的抑制作用。研究表明,以不同浓度的苦杏仁苷作用于BSMC后,在24hrs后开始出现抑制,并且随着浓度的增大抑制作用增强,加药浓度为0.704 mmol/L时抑制率达到50%。
The methods of obtaining bronchus smooth muscle cell (BSMC) were dicussed: digest method.after getting the cells from the wista rat,then 0.05% Trypsin for 1hr and 30mins in 37℃.one week later,cells were full of the base of the bottle.the BSMC were subcultured in DMEM-F12 containing 10% FBS with zobary and spindly smooth membrane.
The Objection of the thesis is to construct green fluorescent protein (pEGFP-N1)- BKβ_1 fusion gene vector for observing the expression and localization of BKβ_1 in HEK-293 cells. the coding region in cDNA was amplified by RT-PCR from rat airway smooth muscle cells and rat bronchia smooth muscle cells.Total RNA is extracted from the 3rd - 5th generation cells,reversely translated,then followed with PCR and recombined into pEGFP-N1 plasmid expressing pEGFP-N1.After identification with restriction endonucleases and sequence analysis,the recombinant plasmid will be transfected into HEK-293 cells with the cationic liposome DOTAP as the transfection reagent.
Amygdalin (D-mandelonitrile-β-D-gentiobioside) is a natural compound with the anti-tussive and anticancer activities.It is decomposed by the action ofβ-D-glucosidase to yield hydrocyanic acid which stimulates the respiratory center reflexively and produces a kind of antitussive and antiasthmatic effects.amygdalin is important in respiratory disease and cardiovascular disease.In this thesis,Amygdalin inhibited BSMC proliferation by 50% after 24hrs.
引文
[1]帅锋利,刘远厚.电导钙激活钾通道β1亚基与心血管疾病.国际检验医学杂志.2006, (5):463-365
[2]刘书宏,曾晓荣,杨艳等.大电导钙激活钾通道及其与高血压的关系.分子生物医学.2006,2(15),897-898 calcium.Neuroscientist,2001,7 (2):166 - 177
[3]洪立立,付玮.大电导钙激活钾通道在平滑肌细胞中的调节机制.国外医学药学分册.2006,3(4):07-30
[4] Gardos,G.(1958).The function of calcium in the potassium permeability of human erythrocytes.Biochim Biophys Acta 30:653– 654
[5] BlatzA.L.& Magleb,K.L.(1986).Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle.Nature 323,718– 720
[6] Srinivas Ghatta a,Deepthi Nimmagadda,Xiaoping Xu,Stephen T.O’Rourke(2006) Large-conductance,calcium-activated potassium channels Structural and functional implications.Pharmacology & Therapeutics110,103– 116
[7] Wallner,M.,Meera,P.& Toro,L. (1996).Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels;an additional transmembrane region at the N terminus. Proc Natl Acad Sci USA 93,14922– 14927
[8] Brenner,R.,Jegla,T. J.,Wickenden Liu,Y. & Aldrich R. W. (2000) Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits,hKCNMB3 and hKCNMB4. BiolChem 275,6453– 6461
[9] Pluznick J.L.,Wei P.,Carmines P.K.& Sansom S.C.(2003).Renal fluid and electrolyte handling in BKCa-beta1-/- mice.Am J Physiol Renal Physiol 284,F1274– F1279
[10] Wei A.,Solaro C.,Lingle C.& Salkoff.L.(1994).Calcium sensitivity of BK-type KCa channels determined by a separable domain.Neuron 13,671–681
[11] Jiang Z,Wallner M,Meera P,et al. Human and rodent Maxi K channel beta2 subunit genes:cloning and characterization.Genomics,1999,55 (1):57267
[12] Dusek L.,Svobodova Z,J anouskova D,et al.Bioaccumulation of mercury in muscle tissue of fish in the Elbe River (Czech Republic):multispecies monitoring study 1991-1996.Ecotoxicol Environ Saf,2005,61 (2):2562267
[13] Stern AH.A revised probabilistic estimate of the maternal met hylmercury intake dose corresponding to a measured cord blood mercury concent ration.Environ Health Perspect,2005,113 ( 2):1552 163
[14] Rice DC.Identification of functional domains affected by develop mental exposure to met hylmercury:Faroe islands and related studies.Neurotoxicology:2000,21(6): 1039-1044
[15] Trasande L,Landrigan PJ,Schechter C.Public health and economic consequences of met hymercury toxicity to the developing brain. Environ Health Perspect,2005,113 (5):590-596
[16] Jaggar J H,Porter VA,Lederer WJ,et al.Calcium sparks in smooth muscle.Am J Physiol Cell Physiol,2000,278 (2):235-256
[17] Amberg GC,Bonev AD,Rossow CF,et al.Modulation of the molecular composition of large conductance,Ca 2 + activated K + channels in vascular smooth muscle during hypertension.J Clin Invest,2003,112 (5):717-724
[18] Nishimaru K,Eghbali M,Lu R,et al.Functional and molecular evidence of Maxi Kchannel beta1 subunit decrease with coronary artery ageing in the rat.J Physiol, 2004,559 (3):849-862
[19] Marijic J,Li Q,Song M,et al.Decreased expression of voltage and Ca 2 + activated K+ channels in coronary smooth muscle during aging.Circ Res,2001,88 (2):210-216
[20] R.Behrens,A.Nolting,F.Reimann,M.Schwarz,R.Waldschutz,O.Pongs.(2000) hKCNMB3 and hKCNMB4,cloning and characterization of two members of the large-conductance calcium-activated potassium channel L subunit family.Federationof European Biochemical Societies.99-106
[21] Latorre R,Oberhauser A,Labarca P,et al.Varieties of calcium - activated potassiumchannels.Ann Rev Physiol,1989,51:385 -399
[22] Wu SN.Largeconductance Ca2 + activated K+ channels:physiological role and pharmacology.Curr Med Chem,2003,10 (8):649 - 661
[23] Jin W.,Sugaya A.,Tsuda T.,Ohguchi H.& Sugaya,E.(2000).Relationship between large conductance calcium-activated potassium channel and bursting activity.Brain Res 860,21– 28
[24] Ruttiger L.,Sausbier M.,Zimmermann U.,Winter H.,Braig C.,Engel J.,et al. (2004).Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.Proc Natl Acad Sci USA 101,12922–12927
[25] Sausbier M.,Hu H.,Arntz C.,Feil S.,Kamm S.,Adelsberger H.,et al. (2004).Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency. Proc Natl Acad Sci USA 101,9474–9478
[26] Pluznick JL,Wei P,Carmines PK,et al.Renal fluid and electrolyte handling in BKCa beta1 mice.Am J Physiol Renal Physiol,2003,284 (6):1274-1279
[27] Zhang Y,Gao YJ,Zuo J,et al. Alteration of arterial smooth muscle potassium channel composition and BKCa current modulation in hypertension.Eur J Pharmacol,2005,514 (223):111-119
[28] Paterno R,Heistad DD,Faraci FM. Functional activity of Ca2 +dependent K+ channels is increased in basilar artery during chronic hypertension.AmJ Physiol,1997,272 (2):H1287-1291
[29] Xu H,Bian X,Watts SW,et al.Activation of vascular BK channel by tempol in DOCA salt hypertensive rats.Hypertension,2005,46 (5):1154-1162
[30] Visser AJ,van Mastrigt R.Intracellular recording of spontaneous electrical activity in human urinary bladder smooth muscle strips.Arch Physiol Biochem,1999,107(3):257-270
[31] Herrera GM,Nelson MT.Differential regulation of SKca and BKchannels by Ca2 + signals from Ca2 + channels and ryanodine receptors in guineapig urinary bladder myocytes.J Physiol,2002,541 (2):483-492
[32] Herrera GM,Heppner TJ,Nelson MT.Voltage dependence of the coupling of Ca2+ sparks to BK(Ca) channels in urinary bladder smooth muscle.AmJ Physiol,2001,280(3):C481-490
[33] Heppner TJ,Bonev AD,Nelson MT.Ca2 +activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle.Am J Physiol,1997,273 (11):C110-117
[34] Herrera GM,Heppner TJ,Nelson MT.Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SKca channels.AmJ Physiol,2000,279 (1):R60-68
[35] Petkov GV,Bonev AD,Heppner TJ,et al.β1Subunit of the Ca2 +activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.J Physio,2001,537:443-452
[36] Buckner SA,Milicic I,Daza AV,et al.Spontaneous phasic activity of the pig urinary bladder smooth muscle:characteristics and sensitivity to potassium channel modulators.Br J Pharmacol,2002,135 (3):6392648
[37]邢国秀,李楠,杨美燕,崔丽钧,王童.天然苦杏仁苷的研究进展.中成药.2003,25(12):107-109
[38]周金黄,王筠默.中药药理学.上海:上海科学技术出版社,1986:219
[39]车文一,金泰日,张良和等.苦杏仁提取物的抗癌作用及其对移植性肝癌小鼠细胞色素P2450含量和谷胱苷肽S2转移酶活性的影响.延边医学院学报,1987,10 (4):223
[40]朱友平,苏中武,李承祜.苦杏仁苷的镇痛作用和无身体依赖性.中国中药杂志,1994,19 (2):105
[2]刘书宏,曾晓荣,杨艳等.大电导钙激活钾通道及其与高血压的关系.分子生物医学.2006,2(15),897-898 calcium.Neuroscientist,2001,7 (2):166 - 177
[3]洪立立,付玮.大电导钙激活钾通道在平滑肌细胞中的调节机制.国外医学药学分册.2006,3(4):07-30
[4] Gardos,G.(1958).The function of calcium in the potassium permeability of human erythrocytes.Biochim Biophys Acta 30:653– 654
[5] BlatzA.L.& Magleb,K.L.(1986).Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle.Nature 323,718– 720
[6] Srinivas Ghatta a,Deepthi Nimmagadda,Xiaoping Xu,Stephen T.O’Rourke(2006) Large-conductance,calcium-activated potassium channels Structural and functional implications.Pharmacology & Therapeutics110,103– 116
[7] Wallner,M.,Meera,P.& Toro,L. (1996).Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels;an additional transmembrane region at the N terminus. Proc Natl Acad Sci USA 93,14922– 14927
[8] Brenner,R.,Jegla,T. J.,Wickenden Liu,Y. & Aldrich R. W. (2000) Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits,hKCNMB3 and hKCNMB4. BiolChem 275,6453– 6461
[9] Pluznick J.L.,Wei P.,Carmines P.K.& Sansom S.C.(2003).Renal fluid and electrolyte handling in BKCa-beta1-/- mice.Am J Physiol Renal Physiol 284,F1274– F1279
[10] Wei A.,Solaro C.,Lingle C.& Salkoff.L.(1994).Calcium sensitivity of BK-type KCa channels determined by a separable domain.Neuron 13,671–681
[11] Jiang Z,Wallner M,Meera P,et al. Human and rodent Maxi K channel beta2 subunit genes:cloning and characterization.Genomics,1999,55 (1):57267
[12] Dusek L.,Svobodova Z,J anouskova D,et al.Bioaccumulation of mercury in muscle tissue of fish in the Elbe River (Czech Republic):multispecies monitoring study 1991-1996.Ecotoxicol Environ Saf,2005,61 (2):2562267
[13] Stern AH.A revised probabilistic estimate of the maternal met hylmercury intake dose corresponding to a measured cord blood mercury concent ration.Environ Health Perspect,2005,113 ( 2):1552 163
[14] Rice DC.Identification of functional domains affected by develop mental exposure to met hylmercury:Faroe islands and related studies.Neurotoxicology:2000,21(6): 1039-1044
[15] Trasande L,Landrigan PJ,Schechter C.Public health and economic consequences of met hymercury toxicity to the developing brain. Environ Health Perspect,2005,113 (5):590-596
[16] Jaggar J H,Porter VA,Lederer WJ,et al.Calcium sparks in smooth muscle.Am J Physiol Cell Physiol,2000,278 (2):235-256
[17] Amberg GC,Bonev AD,Rossow CF,et al.Modulation of the molecular composition of large conductance,Ca 2 + activated K + channels in vascular smooth muscle during hypertension.J Clin Invest,2003,112 (5):717-724
[18] Nishimaru K,Eghbali M,Lu R,et al.Functional and molecular evidence of Maxi Kchannel beta1 subunit decrease with coronary artery ageing in the rat.J Physiol, 2004,559 (3):849-862
[19] Marijic J,Li Q,Song M,et al.Decreased expression of voltage and Ca 2 + activated K+ channels in coronary smooth muscle during aging.Circ Res,2001,88 (2):210-216
[20] R.Behrens,A.Nolting,F.Reimann,M.Schwarz,R.Waldschutz,O.Pongs.(2000) hKCNMB3 and hKCNMB4,cloning and characterization of two members of the large-conductance calcium-activated potassium channel L subunit family.Federationof European Biochemical Societies.99-106
[21] Latorre R,Oberhauser A,Labarca P,et al.Varieties of calcium - activated potassiumchannels.Ann Rev Physiol,1989,51:385 -399
[22] Wu SN.Largeconductance Ca2 + activated K+ channels:physiological role and pharmacology.Curr Med Chem,2003,10 (8):649 - 661
[23] Jin W.,Sugaya A.,Tsuda T.,Ohguchi H.& Sugaya,E.(2000).Relationship between large conductance calcium-activated potassium channel and bursting activity.Brain Res 860,21– 28
[24] Ruttiger L.,Sausbier M.,Zimmermann U.,Winter H.,Braig C.,Engel J.,et al. (2004).Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.Proc Natl Acad Sci USA 101,12922–12927
[25] Sausbier M.,Hu H.,Arntz C.,Feil S.,Kamm S.,Adelsberger H.,et al. (2004).Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency. Proc Natl Acad Sci USA 101,9474–9478
[26] Pluznick JL,Wei P,Carmines PK,et al.Renal fluid and electrolyte handling in BKCa beta1 mice.Am J Physiol Renal Physiol,2003,284 (6):1274-1279
[27] Zhang Y,Gao YJ,Zuo J,et al. Alteration of arterial smooth muscle potassium channel composition and BKCa current modulation in hypertension.Eur J Pharmacol,2005,514 (223):111-119
[28] Paterno R,Heistad DD,Faraci FM. Functional activity of Ca2 +dependent K+ channels is increased in basilar artery during chronic hypertension.AmJ Physiol,1997,272 (2):H1287-1291
[29] Xu H,Bian X,Watts SW,et al.Activation of vascular BK channel by tempol in DOCA salt hypertensive rats.Hypertension,2005,46 (5):1154-1162
[30] Visser AJ,van Mastrigt R.Intracellular recording of spontaneous electrical activity in human urinary bladder smooth muscle strips.Arch Physiol Biochem,1999,107(3):257-270
[31] Herrera GM,Nelson MT.Differential regulation of SKca and BKchannels by Ca2 + signals from Ca2 + channels and ryanodine receptors in guineapig urinary bladder myocytes.J Physiol,2002,541 (2):483-492
[32] Herrera GM,Heppner TJ,Nelson MT.Voltage dependence of the coupling of Ca2+ sparks to BK(Ca) channels in urinary bladder smooth muscle.AmJ Physiol,2001,280(3):C481-490
[33] Heppner TJ,Bonev AD,Nelson MT.Ca2 +activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle.Am J Physiol,1997,273 (11):C110-117
[34] Herrera GM,Heppner TJ,Nelson MT.Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SKca channels.AmJ Physiol,2000,279 (1):R60-68
[35] Petkov GV,Bonev AD,Heppner TJ,et al.β1Subunit of the Ca2 +activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.J Physio,2001,537:443-452
[36] Buckner SA,Milicic I,Daza AV,et al.Spontaneous phasic activity of the pig urinary bladder smooth muscle:characteristics and sensitivity to potassium channel modulators.Br J Pharmacol,2002,135 (3):6392648
[37]邢国秀,李楠,杨美燕,崔丽钧,王童.天然苦杏仁苷的研究进展.中成药.2003,25(12):107-109
[38]周金黄,王筠默.中药药理学.上海:上海科学技术出版社,1986:219
[39]车文一,金泰日,张良和等.苦杏仁提取物的抗癌作用及其对移植性肝癌小鼠细胞色素P2450含量和谷胱苷肽S2转移酶活性的影响.延边医学院学报,1987,10 (4):223
[40]朱友平,苏中武,李承祜.苦杏仁苷的镇痛作用和无身体依赖性.中国中药杂志,1994,19 (2):105