细胞外基质对哮喘模型大鼠气道平滑肌细胞中MAPK、PI3K、PKC表达的影响
|
摘要
前言:气道慢性炎症和气道重塑是支气管哮喘的重要特征,气道平滑肌细胞(airway smooth muscle cells,ASMCs)及细胞外基质(extracellular matrix, ECM)作为气道壁维持气道结构和功能的重要组成部分,二者在哮喘气道炎症和气道重塑中起着重要作用。有研究证实在哮喘病理发展进程中不仅有ASMCs数量的增加和功能的改变,而且有ECM量和组成的改变;增加的ECM沉积在哮喘整个气道壁,不仅参与气道慢性炎症和气道重塑,也可能对ASMCs的各种生物学行为产生重要的调控作用。ECM能调控ASMCs的生存,增殖和迁移等生物学行为,但ECM对哮喘ASMCs功能调控的细胞分子机制尚不清楚。
丝裂原活化蛋白激酶(Mitogen-activated protein kinase ,MAPK)、蛋白激酶C(Protein kinase C ,PKC)、磷脂酰肌醇-3激酶(Phosphatidylinositol 3-Kinase ,PI3K)信号通路是调控哮喘ASMCs增殖的的主要信号途径,但是否参与ECM对哮喘ASMCs的功能调控,目前尚不清楚。本实验将哮喘及对照组大鼠ASMCs种植于涂覆有ECM(纤维连接蛋白、Ⅰ型胶原及层粘蛋白)的培养瓶中,检测各组ASMCs中PI3K、PKC以及MAPK家族中的ERK1/2 mRNA及蛋白的表达情况,了解在哮喘模型大鼠中ECM对MAPK、PI3K、PKC信号分子的影响;以进一步探索ECM是否通过MAPK、PI3K、PKC信号途径对哮喘ASMCs进行功能调控。
目的:观察ECM对哮喘模型大鼠ASMCs中信号分子MAPK(ERK1/2)、PI3K、PKC表达的影响。
方法: 24只雄性wistar大鼠随机分为哮喘组(12只)和对照组(12只) ,建立哮喘模型;培养哮喘及对照组大鼠ASMCs;然后将哮喘组及对照组ASMCs分别种植在已铺ECM(分别为纤维连接蛋白、Ⅰ型胶原及层粘蛋白)的培养瓶中培养;利用实时定量PCR法检测各组ASMCs中ERK1/2、PI3Kp85、PKC-amRNA的表达;Western blot检测各组ASMCs中ERK1/2、PI3Kp85、PKC-a蛋白的表达。
结果: ERK1/2、PI3Kp85、PKC-amRNA及蛋白的表达哮喘组均明显高于对照组(均p<0.01)。哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组的ERK1/2 mRNA相对表达分别是7.262±0.614、3.278±0.037、4.122±0.125,均明显高于哮喘空白组(1.525±0.054)(均p<0.01);哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组PI3Kp85mRNA的相对表达分别是7.626±0.613、9.729±O.682、16.219±0.879,明显高于哮喘空白组(5.931±0.749)(均p<0.01);哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组PKC-amRNA相对表达分别是7.626±0.615、9.927±0.122、8.951±0.237,亦明显高于哮喘空白组(3.478±0.747)(均p<0.01)。对照组ASMCs各组用细胞外基质处理组与未用细胞外基质处理组(空白组)对比基因表达变化不明显(P>0.05)。各组ASMCs中ERK1/2、PKC-α、PI3Kp85的蛋白表达情况与其mRNA表达趋势是一致的,即ERK1/2、PKC-α、PI3Kp85蛋白的表达哮喘纤维粘连蛋白组、层粘蛋白组、Ⅰ型胶原组均高于哮喘空白组(均p<0.01)。
结论: ECM(包括纤维连接蛋白、层粘蛋白及Ⅰ型胶原)使哮喘大鼠ASMCs中ERK1/2、PKC-α、PI3Kp85mRNA及蛋白表达上调; ECM可能通过MAPK、PKC、PI3K信号途径对哮喘ASMCs进行功能调控。
Preface: Airway chronic inflammation and airway reconstitution are importance characters of asthma. As important members of airway wall ,Airway smooth muscle cells and extracellular matrix play important roles in airway chronic inflammation and airway reconstitution of asthma. It is clear that the number and function of airway smooth muscle cell is unlikeliness in asthma to the normal,and the number of extracellular matrix is also distinctness . Extracellular matrix can control the function of Airway smooth muscle cells,but the mechanism is unclear. The primary signal system accelerating the number of ASMCs are Mitogen-activated protein kinase(MAPK), Phosphatidylinositol 3-Kinase(PI3K), Protein kinase C (PKC)signal system.But it is not clear that if extracellular matrix control the function of Airway smooth muscle cells by MAPK,PI3K,PKCsignal system.So we culture the airway smooth cells of asthmatic rats and control rats, seed the ASMCs in flasks which had treaded by extracellular matrix proteinon(conclusion of fironectin,collagen 1,laminin) ,then to delect the gene expression of ERK1/2、PI3K、PKC of the the airway smooth cells by the real time PCR and the protein expression of ERK1/2、PI3Kp85、PKC-aby Western blotting . To observe the influence of extracellular matrix protein on expression of MAPK, PI3K, PKC of airway smooth muscle cells in asthmatic rats.
Objective: To observe the influence of extracellular matrix protein on expression of MAPK, PI3K, PKCof airway smooth muscle cells in asthmatic rats.
Methods: Twenty-four Wistar rats were divided an asthmatic group (n=12)and a control group (n=12); then culture the airway smooth cells. The airway smooth cells were seeded in cuture flasks which had treaded by extracellular matrix proteinon(conclusion of fironectin,collagen 1,laminin) ,then to delect the gene expression of ERK1/2、PI3Kp85、PKC-aof the the airway smooth cells by the real time PCR and the protein expression of ERK1/2、PI3Kp85、PKC-aby Western blotting .
Result: The value of gene expression ofERK1/2、PI3Kp85、PKC-aand protein expression of ERK1/2、PI3Kp85、PKC-ain asthmatic group were higher than the control group (p<0.01) . In asthmatic group, The gene expression relative value of ERK1/2 treated with fironectin , collagen 1,laminin were 7.262±0.614, 3.278±0.037,4.122±0.125;which were higer than asthmatic group untreated with extracellular matrix proteins(1.525±0.054)(p<0.01). The gene expression relative value of PI3Kp85 in asthmatic group treated with fironectin ,collagen 1,laminin were7.626±0.615,9.927±0.122,8.951±0.237;which were higer cpmpared with asthmatic group untreated by extracellular matrix proteins(5.931±0.749)(p<0.01). The gene expression relative value of PKC-a in asthmatic group treated with fironectin ,collagen 1,laminin were7.626±0.615、9.927±0.122、8.951±0.237;which were also higer than those of asthmatic group without treatment(3.478±0.747)(p<0.01). In control group ,The gene expression of ERK1/2、PI3Kp85、PKC-ain ASMCs treated with fironectin ,collagen 1,laminin were indistinctive to that of without treatment(p>0.05).The protein expression of ERK1/2、PI3Kp85、PKC-awere similar to the gene expression in asthmatic group.
Conclusions: Extracellular matrix(including of fironectin,collagenⅠ,laminin can make the gene and protein expression of ERK1/2、PKC-α、PI3Kp85 increase in asthmatic rats ASMCs. extracellular matrix protein may control the function of airway smooth muscle cells by MAPK,PI3K and PKC Signal Transduction System.
丝裂原活化蛋白激酶(Mitogen-activated protein kinase ,MAPK)、蛋白激酶C(Protein kinase C ,PKC)、磷脂酰肌醇-3激酶(Phosphatidylinositol 3-Kinase ,PI3K)信号通路是调控哮喘ASMCs增殖的的主要信号途径,但是否参与ECM对哮喘ASMCs的功能调控,目前尚不清楚。本实验将哮喘及对照组大鼠ASMCs种植于涂覆有ECM(纤维连接蛋白、Ⅰ型胶原及层粘蛋白)的培养瓶中,检测各组ASMCs中PI3K、PKC以及MAPK家族中的ERK1/2 mRNA及蛋白的表达情况,了解在哮喘模型大鼠中ECM对MAPK、PI3K、PKC信号分子的影响;以进一步探索ECM是否通过MAPK、PI3K、PKC信号途径对哮喘ASMCs进行功能调控。
目的:观察ECM对哮喘模型大鼠ASMCs中信号分子MAPK(ERK1/2)、PI3K、PKC表达的影响。
方法: 24只雄性wistar大鼠随机分为哮喘组(12只)和对照组(12只) ,建立哮喘模型;培养哮喘及对照组大鼠ASMCs;然后将哮喘组及对照组ASMCs分别种植在已铺ECM(分别为纤维连接蛋白、Ⅰ型胶原及层粘蛋白)的培养瓶中培养;利用实时定量PCR法检测各组ASMCs中ERK1/2、PI3Kp85、PKC-amRNA的表达;Western blot检测各组ASMCs中ERK1/2、PI3Kp85、PKC-a蛋白的表达。
结果: ERK1/2、PI3Kp85、PKC-amRNA及蛋白的表达哮喘组均明显高于对照组(均p<0.01)。哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组的ERK1/2 mRNA相对表达分别是7.262±0.614、3.278±0.037、4.122±0.125,均明显高于哮喘空白组(1.525±0.054)(均p<0.01);哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组PI3Kp85mRNA的相对表达分别是7.626±0.613、9.729±O.682、16.219±0.879,明显高于哮喘空白组(5.931±0.749)(均p<0.01);哮喘纤维连接蛋白组、层粘蛋白组、Ⅰ型胶原组PKC-amRNA相对表达分别是7.626±0.615、9.927±0.122、8.951±0.237,亦明显高于哮喘空白组(3.478±0.747)(均p<0.01)。对照组ASMCs各组用细胞外基质处理组与未用细胞外基质处理组(空白组)对比基因表达变化不明显(P>0.05)。各组ASMCs中ERK1/2、PKC-α、PI3Kp85的蛋白表达情况与其mRNA表达趋势是一致的,即ERK1/2、PKC-α、PI3Kp85蛋白的表达哮喘纤维粘连蛋白组、层粘蛋白组、Ⅰ型胶原组均高于哮喘空白组(均p<0.01)。
结论: ECM(包括纤维连接蛋白、层粘蛋白及Ⅰ型胶原)使哮喘大鼠ASMCs中ERK1/2、PKC-α、PI3Kp85mRNA及蛋白表达上调; ECM可能通过MAPK、PKC、PI3K信号途径对哮喘ASMCs进行功能调控。
Preface: Airway chronic inflammation and airway reconstitution are importance characters of asthma. As important members of airway wall ,Airway smooth muscle cells and extracellular matrix play important roles in airway chronic inflammation and airway reconstitution of asthma. It is clear that the number and function of airway smooth muscle cell is unlikeliness in asthma to the normal,and the number of extracellular matrix is also distinctness . Extracellular matrix can control the function of Airway smooth muscle cells,but the mechanism is unclear. The primary signal system accelerating the number of ASMCs are Mitogen-activated protein kinase(MAPK), Phosphatidylinositol 3-Kinase(PI3K), Protein kinase C (PKC)signal system.But it is not clear that if extracellular matrix control the function of Airway smooth muscle cells by MAPK,PI3K,PKCsignal system.So we culture the airway smooth cells of asthmatic rats and control rats, seed the ASMCs in flasks which had treaded by extracellular matrix proteinon(conclusion of fironectin,collagen 1,laminin) ,then to delect the gene expression of ERK1/2、PI3K、PKC of the the airway smooth cells by the real time PCR and the protein expression of ERK1/2、PI3Kp85、PKC-aby Western blotting . To observe the influence of extracellular matrix protein on expression of MAPK, PI3K, PKC of airway smooth muscle cells in asthmatic rats.
Objective: To observe the influence of extracellular matrix protein on expression of MAPK, PI3K, PKCof airway smooth muscle cells in asthmatic rats.
Methods: Twenty-four Wistar rats were divided an asthmatic group (n=12)and a control group (n=12); then culture the airway smooth cells. The airway smooth cells were seeded in cuture flasks which had treaded by extracellular matrix proteinon(conclusion of fironectin,collagen 1,laminin) ,then to delect the gene expression of ERK1/2、PI3Kp85、PKC-aof the the airway smooth cells by the real time PCR and the protein expression of ERK1/2、PI3Kp85、PKC-aby Western blotting .
Result: The value of gene expression ofERK1/2、PI3Kp85、PKC-aand protein expression of ERK1/2、PI3Kp85、PKC-ain asthmatic group were higher than the control group (p<0.01) . In asthmatic group, The gene expression relative value of ERK1/2 treated with fironectin , collagen 1,laminin were 7.262±0.614, 3.278±0.037,4.122±0.125;which were higer than asthmatic group untreated with extracellular matrix proteins(1.525±0.054)(p<0.01). The gene expression relative value of PI3Kp85 in asthmatic group treated with fironectin ,collagen 1,laminin were7.626±0.615,9.927±0.122,8.951±0.237;which were higer cpmpared with asthmatic group untreated by extracellular matrix proteins(5.931±0.749)(p<0.01). The gene expression relative value of PKC-a in asthmatic group treated with fironectin ,collagen 1,laminin were7.626±0.615、9.927±0.122、8.951±0.237;which were also higer than those of asthmatic group without treatment(3.478±0.747)(p<0.01). In control group ,The gene expression of ERK1/2、PI3Kp85、PKC-ain ASMCs treated with fironectin ,collagen 1,laminin were indistinctive to that of without treatment(p>0.05).The protein expression of ERK1/2、PI3Kp85、PKC-awere similar to the gene expression in asthmatic group.
Conclusions: Extracellular matrix(including of fironectin,collagenⅠ,laminin can make the gene and protein expression of ERK1/2、PKC-α、PI3Kp85 increase in asthmatic rats ASMCs. extracellular matrix protein may control the function of airway smooth muscle cells by MAPK,PI3K and PKC Signal Transduction System.
引文
1 Hirst SJ. Regulation of airway smooth muscle cell immunomodulatory function: role in asthma. Respir Physiol Neurobiol JT - Respiratory physiology & neurobiology, 2003,137(2-3):309-26.
2 Johnson PR, Burgess JK, Underwood PA, et al. Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism. J Allergy Clin Immunol JT - The Journal of allergy and clinical immunology, 2004,113(4):690-6.
3 Chan V, Burgess JK, Ratoff JC, et al. Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells. Am J Respir Crit Care Med, 2006,174(4):379-85.
4 Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol, 2003,137(2-3):295-308.
5 Tang YL, Hu CP, Feng JT, et al. [Regulation effect of nerve growth factor on Ras-MAPK signal transduction pathway in neurogenic inflammation of asthma]. Zhong Nan Da Xue Xue Bao Yi Xue Ban, 2006,31(3):319-25.
6 XU Shuyun许淑云. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chinese Medical Journal, 2004,117 ( 1) : 30236:117 ( 1) : 30236.
7 Nguyen TT, Ward JP, Hirst SJ. beta1-Integrins mediate enhancement of airway smooth muscle proliferation by collagen and fibronectin. Am J Respir Crit Care Med, 2005,171(3):217-23.
8 Hirst SJ. Regulation of airway smooth muscle cell immunomodulatory function: role in asthma. Respir Physiol Neurobiol, 2003,137(2-3):309-26.
9 Panettieri RA Jr. Airway smooth muscle: immunomodulatory cells that modulate airway remodeling?. Respir Physiol Neurobiol, 2003,137(2-3):277-93.
10 Parameswaran K, Radford K, Zuo J, et al. Extracellular matrix regulates human airway smooth muscle cell migration. Eur Respir J, 2004,24(4):545-51.
11 Vignola AM, Mirabella F, Costanzo G, et al. Airway remodeling in asthma. Chest, 2003,123(3 Suppl):417S-22S.
12 Bonacci JV, Harris T, Stewart AG. Impact of extracellular matrix and strain on proliferation of bovine airway smooth muscle. Clin Exp Pharmacol Physiol, 2003,30(5-6):324-8.
13 Black JL, Burgess JK, Johnson PR. Airway smooth muscle--its relationship to the extracellular matrix. Respir Physiol Neurobiol, 2003,137(2-3):339-46.
14 Farias E, Lu M, Li X, et al. Integrin alpha8beta1-fibronectin interactions promote cell survival via PI3 kinase pathway. Biochem Biophys Res Commun, 2005,329(1):305-11.
15 Jadlowiec J, Koch H, Zhang X, et al. Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway. J Biol Chem, 2004,279(51):53323-30.
16 Buensuceso CS, Obergfell A, Soriani A, et al. Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta. J Biol Chem, 2005,280(1):644-53.
1 Cooray S. The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival. J Gen Virol, 2004,85(Pt 5):1065-76.
2 Merlot S, Firtel RA. Leading the way: Directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J Cell Sci, 2003,116(Pt 17):3471-8.
3王芳,曹西南.蛋白激酶Cε的结构特点和生理、病理作用.国外医学.临床生物化学与检验学分册, 2005,(05).
4 Song YF, Xu LM, Hong JH. [Relationship between transforming growth factor-betal and fibrosis: its c-jun N-terminal kinase and p38 mitogen-activated protein kinase pathways and inhibitors]. Zhong Xi Yi Jie He Xue Bao, 2004,2(5):382-4.
5 Dorion S, Landry J. Activation of the mitogen-activated protein kinase pathways by heat shock. Cell Stress Chaperones, 2002,7(2):200-6.
6 Ito K, Caramori G, Adcock IM. Therapeutic potential of phosphatidylinositol
3-kinase inhibitors in inflammatory respiratory disease. J Pharmacol Exp Ther, 2007,321(1):1-8.
7 Xiong W, Xu Y, Zhang Z, et al. Nuclear factor-kappa B in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model. Chin Med J (Engl), 2002,115(5):685-9.
8张维溪. MAPK信号转导途径及其在支气管哮喘中的作用.国外医学.呼吸系统分册, 2002,(05).
9高伟良,邱晨.介导气道平滑肌细胞增殖的信号转导途径.国外医学.内科学分册, 2005,(04).
10 Xu SY, Xu YJ, Zhang ZX, et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl), 2004,117(1):30-6.
11许淑云,徐永健,张珍祥,等.蛋白激酶C在支气管哮喘模型大鼠气道平滑肌细胞增殖中的信号转导机制研究.中华结核和呼吸杂志, 2003,(12).
12 Newton R, Holden N. Inhibitors of p38 mitogen-activated protein kinase: potential as anti-inflammatory agents in asthma?. BioDrugs, 2003,17(2):113-29.
13 Chialda L, Zhang M, Brune K, et al. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia. Respir Res, 2005,6:36.
14 Lee KS, Lee HK, Hayflick JS, et al. Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J, 2006,20(3):455-65.
1 Marino JH, Cook P,Miller KS. Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR. J Immunol Methods, 2003,283:291-306.
2 Suzuki N, Yoshida A,Nakano Y. Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR. Clin Med Res, 2005,3:176-85.
3 Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 2000,25:169-93.
4 Romanowski T, Markiewicz A, Bednarz N, et al. [Housekeeping genes as a reference in quantitative real-time RT-PCR]. Postepy Hig Med Dosw (Online), 2007,61:500-10.
5陈凤花,王琳,胡丽华.实时荧光定量RT-PCR内参基因的选择.临床检验杂志, 2005,14:89-91.
6 Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 2001,29:e45.
7 Sellars MJ, Vuocolo T, Leeton LA, et al. Real-time RT-PCR quantification of Kuruma shrimp transcripts: a comparison of relative and absolute quantification procedures. J Biotechnol, 2007,129:391-9.
8 Espy MJ, Uhl JR, Sloan LM, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev, 2006,19:165-256.
9 Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ, 2005,29:151-9.
10 Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin Chem, 2002,48:1178-85.
11 Kao CL, King CC, Chao DY, et al. Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health. J Microbiol Immunol Infect, 2005,38:5-16.
12 Dvorak Z, Pascussi JM,Modriansky M. Approaches to messenger RNA detection - comparison of methods. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2003,147:131-5.
13 Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002,29:23-39.
2 Johnson PR, Burgess JK, Underwood PA, et al. Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism. J Allergy Clin Immunol JT - The Journal of allergy and clinical immunology, 2004,113(4):690-6.
3 Chan V, Burgess JK, Ratoff JC, et al. Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells. Am J Respir Crit Care Med, 2006,174(4):379-85.
4 Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol, 2003,137(2-3):295-308.
5 Tang YL, Hu CP, Feng JT, et al. [Regulation effect of nerve growth factor on Ras-MAPK signal transduction pathway in neurogenic inflammation of asthma]. Zhong Nan Da Xue Xue Bao Yi Xue Ban, 2006,31(3):319-25.
6 XU Shuyun许淑云. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chinese Medical Journal, 2004,117 ( 1) : 30236:117 ( 1) : 30236.
7 Nguyen TT, Ward JP, Hirst SJ. beta1-Integrins mediate enhancement of airway smooth muscle proliferation by collagen and fibronectin. Am J Respir Crit Care Med, 2005,171(3):217-23.
8 Hirst SJ. Regulation of airway smooth muscle cell immunomodulatory function: role in asthma. Respir Physiol Neurobiol, 2003,137(2-3):309-26.
9 Panettieri RA Jr. Airway smooth muscle: immunomodulatory cells that modulate airway remodeling?. Respir Physiol Neurobiol, 2003,137(2-3):277-93.
10 Parameswaran K, Radford K, Zuo J, et al. Extracellular matrix regulates human airway smooth muscle cell migration. Eur Respir J, 2004,24(4):545-51.
11 Vignola AM, Mirabella F, Costanzo G, et al. Airway remodeling in asthma. Chest, 2003,123(3 Suppl):417S-22S.
12 Bonacci JV, Harris T, Stewart AG. Impact of extracellular matrix and strain on proliferation of bovine airway smooth muscle. Clin Exp Pharmacol Physiol, 2003,30(5-6):324-8.
13 Black JL, Burgess JK, Johnson PR. Airway smooth muscle--its relationship to the extracellular matrix. Respir Physiol Neurobiol, 2003,137(2-3):339-46.
14 Farias E, Lu M, Li X, et al. Integrin alpha8beta1-fibronectin interactions promote cell survival via PI3 kinase pathway. Biochem Biophys Res Commun, 2005,329(1):305-11.
15 Jadlowiec J, Koch H, Zhang X, et al. Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway. J Biol Chem, 2004,279(51):53323-30.
16 Buensuceso CS, Obergfell A, Soriani A, et al. Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta. J Biol Chem, 2005,280(1):644-53.
1 Cooray S. The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival. J Gen Virol, 2004,85(Pt 5):1065-76.
2 Merlot S, Firtel RA. Leading the way: Directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J Cell Sci, 2003,116(Pt 17):3471-8.
3王芳,曹西南.蛋白激酶Cε的结构特点和生理、病理作用.国外医学.临床生物化学与检验学分册, 2005,(05).
4 Song YF, Xu LM, Hong JH. [Relationship between transforming growth factor-betal and fibrosis: its c-jun N-terminal kinase and p38 mitogen-activated protein kinase pathways and inhibitors]. Zhong Xi Yi Jie He Xue Bao, 2004,2(5):382-4.
5 Dorion S, Landry J. Activation of the mitogen-activated protein kinase pathways by heat shock. Cell Stress Chaperones, 2002,7(2):200-6.
6 Ito K, Caramori G, Adcock IM. Therapeutic potential of phosphatidylinositol
3-kinase inhibitors in inflammatory respiratory disease. J Pharmacol Exp Ther, 2007,321(1):1-8.
7 Xiong W, Xu Y, Zhang Z, et al. Nuclear factor-kappa B in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model. Chin Med J (Engl), 2002,115(5):685-9.
8张维溪. MAPK信号转导途径及其在支气管哮喘中的作用.国外医学.呼吸系统分册, 2002,(05).
9高伟良,邱晨.介导气道平滑肌细胞增殖的信号转导途径.国外医学.内科学分册, 2005,(04).
10 Xu SY, Xu YJ, Zhang ZX, et al. Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation. Chin Med J (Engl), 2004,117(1):30-6.
11许淑云,徐永健,张珍祥,等.蛋白激酶C在支气管哮喘模型大鼠气道平滑肌细胞增殖中的信号转导机制研究.中华结核和呼吸杂志, 2003,(12).
12 Newton R, Holden N. Inhibitors of p38 mitogen-activated protein kinase: potential as anti-inflammatory agents in asthma?. BioDrugs, 2003,17(2):113-29.
13 Chialda L, Zhang M, Brune K, et al. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia. Respir Res, 2005,6:36.
14 Lee KS, Lee HK, Hayflick JS, et al. Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J, 2006,20(3):455-65.
1 Marino JH, Cook P,Miller KS. Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR. J Immunol Methods, 2003,283:291-306.
2 Suzuki N, Yoshida A,Nakano Y. Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR. Clin Med Res, 2005,3:176-85.
3 Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 2000,25:169-93.
4 Romanowski T, Markiewicz A, Bednarz N, et al. [Housekeeping genes as a reference in quantitative real-time RT-PCR]. Postepy Hig Med Dosw (Online), 2007,61:500-10.
5陈凤花,王琳,胡丽华.实时荧光定量RT-PCR内参基因的选择.临床检验杂志, 2005,14:89-91.
6 Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 2001,29:e45.
7 Sellars MJ, Vuocolo T, Leeton LA, et al. Real-time RT-PCR quantification of Kuruma shrimp transcripts: a comparison of relative and absolute quantification procedures. J Biotechnol, 2007,129:391-9.
8 Espy MJ, Uhl JR, Sloan LM, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev, 2006,19:165-256.
9 Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ, 2005,29:151-9.
10 Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin Chem, 2002,48:1178-85.
11 Kao CL, King CC, Chao DY, et al. Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health. J Microbiol Immunol Infect, 2005,38:5-16.
12 Dvorak Z, Pascussi JM,Modriansky M. Approaches to messenger RNA detection - comparison of methods. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2003,147:131-5.
13 Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol, 2002,29:23-39.