【作者】 杜春玲；

【导师】 徐永健；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 哮喘是一种以气道高反应性,气道炎症和气道重建为特征的慢性疾病,目前患病率显著性增高[1],并且被认为是丧失劳动能力、医疗消费和可预防性死亡的主要原因之一[2]。到目前为止许多研究机构已经证实:在慢性哮喘,气道平滑肌在诱导气道重建中起着主要作用[3-6]。气道平滑肌细胞的反常增殖导致了气道重建和气道高反应性,但是其具体的机制目前还不很清楚。蛋白激酶C(PKC)是一丝氨酸-苏氨酸激酶家族,它包括三种类型的异构体。传统型(α, ?1, ?2,和γ)可被钙、佛波酯和磷脂酰丝氨酸所激活;新型(δ,ε,η,θ, andμ)对钙不敏感,但可被佛波酯和磷脂酰丝氨酸所激活;不典型型(ζandτ/λ)对钙和佛波酯不敏感,但可被磷脂酰丝氨酸激活。人气道平滑肌细胞表达PKCα, ?1, ?2,δ,ε, andζ[7]。不同的异构体的体外激酶活性特征以及组织分布、亚细胞结构是有差别的。这些异构体参与信号通路,调节细胞增殖、分化、凋亡、迁徙和粘附[8-10]。许多研究者已经证明:PKC的激活是ASMCs增殖的必需事件[11-15],并且,不同的PKC异构体在调节细胞增殖中起着不同的作用。在NIH3T3细胞,PKCε是一强有力的生长刺激因素,而PKCα和δ抑制生长[16]。在A7r5血管平滑肌细胞[17],毛细血管内皮细胞[18]和鼠结肠上皮细胞[19],PKCδ也抑制细胞周期进展。在血管平滑肌细胞,PKCδ的过度表达抑制G1期周期蛋白的表达[17],这与PKCδ控制周期蛋白D1的转录的假设一致。在人,牛和鼠气道平滑肌细胞,PKCδ负性调节cyclinD1的转录[20]。我们实验室已经证实:PKCα参与哮喘血清被动致敏的人气道平滑肌细胞增殖[21]。然而,PKCα调节的下游机制目前还不清楚。细胞周期进展是细胞增殖的的基本事件,目前已经有一些关于ASMCs中调节特殊细胞周期蛋白表达的信号转导通路的研究[22]。cyclinD(D1,D2和D3)是哺乳动物细胞G1期进展的关键调节蛋白,cyclinD1是许多生长刺激信号通路的靶基因[23,24]。那么,在哮喘人气道平滑肌细胞,cyclinD1是否起关键作用?PKCα是否可通过调节cyclinD1表达来影响ASMC增殖?为此,我们利用哮喘血清被动致敏的细胞模型来研究这些问题。研究内容1.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs)(B组),以10%非哮喘者血清为对照(A组)。构建cyclinD1正、反义真核表达载体质粒并转染干预HASMC。用RT-PCR方法以及Western Blot方法检测cyclinD1的mRNA及蛋白表达水平,用流式细胞术、四甲基偶氮唑盐(MTT)法及增殖细胞核抗原(PCNA)免疫细胞化学技术检测HASMC增殖。2.生长融合、同步后的HASMCs用哮喘血清致敏后,分为对照组,PMA组和PMA+Go6976组。干预24小时后,用3H-TdR掺入法和MTT法检测细胞增殖;用碘化丙啶单染法流式细胞术分析细胞周期;用RT-PCR和western blotting检测cyclinD1mRNA和蛋白的表达。进一步,我们还通过转染重组的反义cyclinD1质粒(PCDNA3.1(+)/as-cyclinD1)来降低cyclinD1基因的表达后,检测PKCα对HASMCs增殖的影响。3.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和特异性阻断剂U0126抑制PKCα和ERK的表达,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1和P21cip1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。4.用10%哮喘患者血清被动致敏人气道平滑肌细胞(HASMCs),予以PKC激活剂PMA刺激。分别以反义寡核苷酸(PKCα-asODN)和PDTC抑制PKCα表达和NF-κB活化。用凝胶电泳迁移率改变试验(EMSA)检测HASMCs中NF-κB的活性,用RT-PCR方法及Western Blot方法检测干预前后cyclinD1的mRNA及蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MTT)法检测HASMCs增殖。结果1.正常血清对照组细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(10.52±1.50)%、0.303±0.024和(39.4±8.53)%;哮喘血清组(B0组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳率分别为(15.94±2.13)%、0.431±0.047和(53.2±7.5)%,均较对照血清组(A组)明显增加(n=5,P<0.05);转染重组cyclinD1正义质粒组(B2组)细胞S期细胞比例、MTT吸光度值(A值)、PCNA表达阳性分别为(26.10±2.16)%、0.591±0.042和(84.2±5.9)%,均较哮喘血清组(B0组)明显增加(n=5,P<0.05);而转染反义质粒组(B3组)上述结果分别为(6.96±1.25)%、0.220±0.027和(29.8±8.2)%,均较哮喘血清组(B0组)明显下降(n=5,P<0.05)。2. PMA激活PKCα后上调cyclinD1表达和细胞增殖。而用Go6976特异性抑制PKCα活性后,这种促cyclinD1表达和细胞增殖的作用显著性下降。相反, PMA和Go6976对周期蛋白依赖性激酶4(CDK4)的表达无明显影响。另外,转染反义cyclinD1质粒后消除了PMA诱导的G1/S期进展和HASMCs增殖效应。3. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,ERK1/2,p-ERK1/2蛋白水平增高,cyclin D1、P21cip1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞抑制p-PKCα表达后,ERK1/2,p-ERK1/2表达明显减弱,cyclin D1、P21cip1表达也明显下降,HASMCs的增殖减弱(P<0.05,n=4).;用U0126抑制ERK1/2表达后,PMA刺激下p-PKCα水平仍然明显增高,但cyclin D1、P21cip1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4).。4. PMA刺激后磷酸化PKCα(p-PKCα)水平增高,NF-κB-DNA结合活性增强,cyclin D1表达明显增强, HASMCs的增殖增强;而反义PKCα寡核苷酸转入细胞特异性地抑制PΚCα表达后,p-PKCα水平下降,NF-κB-DNA结合活性明显减弱,cyclin D1也明显下降,HASMCs的增殖减弱(P<0.05,n=4);用PDTC抑制NF-κB活性后,PMA刺激下p-PΚCα水平仍然明显增高,但cyclin D1的表达明显下降,HASMCs的增殖减弱(P<0.05,n=4)。结论1. cyclinD1促进哮喘血清被动致敏的HASMC的增殖。cyclinD1可能是哮喘HASMC增殖的重要调控蛋白。2. PKCα可通过上调cyclinD1表达而促进哮喘血清被动致敏的人气道平滑肌细胞增殖。3. ERK1/2是PKCα的下游信号分子,PKCα-ERK1/2级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1、P21cip1的表达上调及细胞增殖。4. NF-κB是PKCα的下游信号分子,PΚCα- NF-κB级联参与了PMA所诱导的哮喘血清被动致敏的人气道平滑细胞cyclin D1的表达上调及细胞增殖。综上所述,上述结果表明:PKCα、cyclinD1参与了哮喘HASMC的增殖调控,并存在PKCα-ERK1/2-cyclinD1、PΚCα-NF-κB-cyclinD1信号转导途径,从不同环节阻断这一信号途径对于针对性地抑制哮喘气道平滑肌增殖、减轻气道重建可能具有积极意义。这为研究哮喘气道重建的发病机理提供新的理论,也为将来治疗难治性哮喘提供一定的新思路。更多还原

【Abstract】 Asthma, a chronic disease characterized by airway hyperreactivity, inflammation, and remodeling, has increased dramatically in the prevalence [1] and is now recongnized as a major cause of disability, medical expense, and preventable death [2]. So far, many researches have confirmed a prominent role of airway smooth muscle (ASM) in the induction of airway remodeling in chronic asthma [3-6]. The abnormal proliferation of ASMC contributes to airway remodeling and hyper-reactivity characteristic of asthma. However, the underlying mechanism remains unclear.Protein kinase C (PKC) is a family of serine/threonine kinases that include three types of isoenzymes. The conventional isoforms (α, ?1, ?2, andγ) are activated by calcium, phorbol esters, and phosphatidylserine, whereas the novel isoforms (δ,ε,η,θ, andμ) are calcium-insensitive and activated by phorbol esters and phosphatidylserine. Theatypical isoforms (ζandτ/λ) are calcium- and phorbol ester–insensitive and activated by phosphatidylserine. PKCα, ?1, ?2,δ,ε, andζare expressed in human tracheal myocytes [7]. Differences between isoforms have been reported in tissue distribution and subcellular localization as well as in vitro kinase -activity characteristics. These isoforms are involved in the signaling pathways that regulate cell proliferation, differentiation, apoptosis, motility and adhesion [8-10].Many reports have demonstrated that activation of PKC is a necessary event for proliferation of ASMCs [11-15], and also different PKC isoforms may have distinct roles in the regulation of cell proliferation. In NIH3T3 cells, PKCεis a powerful growth stimulus, whereas PKCαandδinhibit growth [16]. PKCδalso inhibits cell cycle progression in A7r5 vascular smooth muscle cells [17], capillary endothelial cells [18], and rat colonic epithelial cells [19]. In vascular smooth muscle cells, overexpression of PKCδsuppressed G1 cyclin expression [17], consistent with the hypothesis that cyclin D1 expression is under the transcriptional control of PKCδ. In human, bovine, and rat tracheal smooth muscle cells, PKCδnegtively regulates cyclinD1 transcription [20]. Our laboratory has demonstrated that PKCαmay contribute to proliferation of asthmatic serum passively sensitized human airway smooth muscle cells [21].However, the downstream mechanism of PKCαregulation is unknown now. Progression of ASMC through the cell cycle is a fundamental event of cell proliferation. Recent studies have examined the signal transduction pathways that regulate specific cell cycle protein expression in ASM cells [22]. D-type cyclins (cyclinD1, D2, and D3) have been found to be the key regulators of G1 progression in mammalian cells. The cyclinD1 is one of the major targets for several growth stimulatory signaling pathways [23, 24].However, whether CyclinD1 play a critical role in regulating the HASMCs proliferation in asthma and whether PKCαregulates the proliferation of ASMC via cyclinD expression remains unknown. Therefore, in the present study we examined the possible roles of cyclin D1 andαisoforms of PKC, using human atopic asthmatic serum passively sensitized HASMCs as a model system.Methods1. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients (group B), with non-asthmatic human serum treated HASMCs as the control (group A). The eukaryotic expression plasmids of sense and antisense cyclinD1 gene were constructed and transfected into HASMCs. The levels of cyclinD1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and proliferation cell nuclear antigen (PCNA) immunocytochemistry staining respectively.2. Confluent and synchronized HASMCs were treated with atopic asthmatic serum in the absence and presence of PMA for 24h, or pretreated 30min with Go6976 followed by PMA stimulation for 24h. Thereafter, the proliferation was measured by [3H]-thymidine incorporation and MTT. The cell cycle was analyzed by flow cytometry with propidium iodide staining. The cyclinD1 mRNA and protein expression was measured by RT-PCR and Western blotting. Further, we assessed the role of cyclinD1 in PKCαinduced HASMCs proliferation by cyclinD1 gene knock down using antisense cyclinD1 recombinant (PCDNA3.1(+)/ as-cyclinD1) transfection.3. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, stimulated with PKC activator PMA. The expression of PKCαand ERK1/2 were inhibited by transfected with PKCαantisense Oligodeoxynucleotides (PKCα-asODN) and treated with MAP Kinase Kinase (MEK) inhibitor U0126 respectively. Before and after this treatment, the levels of cyclinD1 and P21cip1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis and MTT colorimetric assay.4. HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, stimulated with PΚC activator PMA. The expression of PΚCαand NF-κB activity were inhibited by transfected with PΚCαantisense Oligodeoxynucleotides (PΚCα-asODN) and treated with PDTC respectively. Before and after this treatment, the NF-κB activity of HASMCs was analyzed by Electrophoretic mobility gel shift assay (EMSA). The levels of cyclinD1 mRNA and protein were detected by RT-PCR and western blot analysis respectively. The proliferation of HASMCs was examined by cell cycle analysis and MTT colorimetric assay.Results1. In non-asthmatic serum treated HASMCs (group A), the percentage of S phase, absorbance value (A value) and the positive expression rate of PCNA were (10.52±1.50)%、0.303±0.024 and (39.4±8.53)% respectively. In asthmatic serum treated HASMCs (group B0), the percentage of S phase, absorbance value (A value) and the positive expression rate of PCNA were (15.94±2.13)%, 0.431±0.047and (53.2±7.5)% respectively. They were significantly increased compared with those of group A (n=5, P<0.05). In HASMCs transfected with sense cyclinD1 recombinant plasmid (group B2), the above figures were (26.10±2.16)%, 0.591±0.042 and(84.2±5.9)% respectively. They were significantly increased compared with those of group B0 (n=5, P<0.05). In HASMCs transfected with antisense cyclinD1 recombinant plasmid (group B3),the above figures were (6.96±1.25)%,0.220±0.027and (29.8±8.2)% respectively. They were significantly decreased compared with those of group B0 (n=5, P<0.05).2. Activation of PKCαwith PMA up-regulated cyclinD1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly inhibited by specific inhibition of PKCαwith Go6976. In contrast, administration of PMA and Go6976 had no effect on cyclin-dependent kinase 4(CDK4) expressions. In addition, we showed that transfection with PCDNA3.1(+)/antisense-cyclinD1 abolished PMA-induced G1/S progression and HASMCs proliferation.3. After stimulated with PMA, the levels of p- PKCαand ERK1/2,p-ERK1/2 increased, the expression of cyclin D1、P21cip1 and cells proliferation enhanced compared with those of the control group(P<0.05,n=4). After transfected with PKCα-asODN, the level of p-PKCαdecreased, the levels of ERK1/2,p-ERK1/2 decreased correspondingly, and the expression of cyclin D1、P21cip1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4). After administration of U0126 , the level of p-PKCαincreased but the levels of ERK1/2,p-ERK1/2 decreased, the expression of cyclin D1、P21cip1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4).4. After stimulated with PMA, the levels of phosphorylated (p-PΚCα) and NF-κB activity increased, the expression of cyclin D1 and cells proliferation enhanced compared with those of the control group(P<0.05,n=4). After transfected with PΚCα-asODN, the level of p-PΚCαdecreased, the NF-κB activity decreased correspondingly, and the expression of cyclin D1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4). After administration of PDTC , the level of p-PΚCα increased but the NF-κB activity decreased, the expression of cyclin D1 and cells proliferation reduced compared with those of the PMA treated alone group (P<0.05,n=4).Conclusions1. CyclinD1 contributes to the proliferation in in passively sensitized HASMCs. CyclinD1 may play a critical role in regulating the HASMCs proliferation in asthma.2. Protein kinase Cαpromoted the proliferation of atopic asthmatic sensitized human airway smooth muscle cells by up-regulating cyclin D1 expression3. ERK1/2 is one of downstream regulator of PKCα. PKCα-ERK1/2 cascade is involved in the PMA induced up-regulation of cyclinD1 and P21cip1 and proliferation in atopic asthmatic sensitized HASMCs4. NF-κB is one of downstream regulator of PΚCα. PΚCα-NF-κB cascade is involved in the PMA induced up-regulation of cyclinD1 and proliferation in atopic asthmatic sensitized HASMCsIn summary, all these findings indicated that PKC and CyclinD1 might take part in the proliferation of asthmatic HASMCs, in which there is PKCα-ERK1/2-cyclinD1、PΚCα-NF-κB-cyclinD1 signal pathway. To block this signal transduction pathway on various points is significant to suppress asthmatic HASMCs proliferation and relieve airway remodeling.The results should be helpful for further providing the novel clue for further elucidating the pathogenesis in asthma and finding the treatment in severe asthma更多还原