【作者】 王正艳；

【导师】 刘瑾；

【作者基本信息】 华中科技大学 ， 呼吸内科， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原