【作者】 向敏；

【导师】 徐永健；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 第一部分蛋白激酶Cα和周期蛋白D1在吸烟患或不患慢性阻塞性肺疾病患者肺血管中的表达变化目的检测蛋白激酶Cα(PKCα)和周期蛋白D1 (cyclinD1)在吸烟患或不患慢性阻塞性肺疾病(COPD)患者肺血管中的表达变化并探讨两者在吸烟导致的肺血管重构中的作用。方法将35例因肺癌而行肺叶切除术患者的外周肺组织标本分为三组：不吸烟肺功能正常组(正常组)10例,吸烟肺功能正常组(吸烟组)14例,吸烟伴COPD组(COPD组)11例。采用苏木精-伊红(HE)染色观察肺小动脉形态学改变,应用免疫组织化学染色法检测肺小动脉平滑肌细胞中α-平滑肌肌动蛋白(α-SMA)、增殖细胞核抗原(PCNA)、PKCα和cyclinD1蛋白表达,以PCNA阳性细胞百分率表示平滑肌细胞增殖指数(PI),采用实时荧光PCR法检测肺小动脉平滑肌细胞中]PKCa mRNA、cyclin D1 mRNA的表达。结果(1)肺小动脉血管壁面积占血管总面积的百分比值(WA%)在吸烟组和COPD组均高于正常组。(2)吸烟组和COPD组PI均高于正常组(P<0.01)。(3)吸烟组、COPD组PKCa蛋白和cyclinD1蛋白表达吸光度(A)值较正常组均显著升高(P<0.01)。(4)吸烟组、COPD组PKCa mRNA和cyclinD1 mRNA表达水平较正常组均显著升高(P<0.01)。(5)PKCa蛋白表达水平分别与WA%、PI呈正相关(P<0.01),cyclinD1蛋白表达水平分别与WA%、PI呈正相关(P<0.01),PKCa与cyclin D1在蛋白和mRNA表达水平均呈明显正相关(P<0.01)。结论在吸烟者及早期COPD患者肺血管中PKCa和cyclinD1表达均显著上调,二者可能参与肺血管重构中肺动脉平滑肌细胞的异常增殖。第二部分周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用目的本文旨在探讨周期蛋白D1(cyclinD1)在香烟烟雾提取物(cigarette smoke extract,CSE)所致人肺动脉平滑肌细胞(HPASMCs)增殖中的作用。方法用不同浓度的CSE(1%,2.5%,5%,10%,20%)干预同步化的HPASMCs。构建反义cyclin D1基因真核表达载体(pIRES2-EGFP-ascyclin D1),采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1导入正常HPASMCs后,分别进行CSE(5%)干预。细胞随机分为6组：对照组、空载体组、反义cyclinD1组、5%CSE组、空载体+5%CSE组、反义cyclin D1+5% CSE组。用台盼蓝染色法检测不同浓度CSE干预的HPASMCs活细胞率,采用四甲基偶氮唑盐比色法(MTT)、流式细胞术、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力,用实时荧光RT-PCR和Western blot法分别检测cyclin D1 mRNA和蛋白的表达。结果低浓度的CSE(1%,2.5%,5%,10%)促进HPASMCs增殖,且浓度5%时细胞增殖能力最强,高浓度的CSE(20%)对HPASMCs具有毒性作用,抑制细胞增殖。反义cyclin D1基因真核表达载体pIRES2-EGFP-ascyclin D1成功构建,并成功转染入HPASMCs,转染后HPASMCs中cyclin D1的mRNA和蛋白表达水平较对照组均显著下降(P<0.05)。与对照组比较,5%CSE组cyclin D1的nRNA和蛋白表达水平均明显升高(P<0.05),细胞增殖能力显著增强(P<0.05)。与5%CSE组比较,反义cyclin D1+5% CSE组cyclin D1 mRNA和蛋白表达水平均明显下降(P<0.05),细胞增殖能力显著降低(P<0.05)。结论CSE可通过上调cyclin D1表达促进HPASMCs增殖,反义cyclin D1基因真核表达载体可抑制CSE介导的HPASMCs增殖,提示cyclin D1在CSE促进HPASMCs增殖中发挥重要的正向调控作用。第三部分蛋白激酶Cα和周期蛋白D1在香烟烟雾提取物促进人肺动脉平滑肌细胞增殖中的作用和关系目的研究蛋白激酶Cα(PKCα)和周期蛋白D1(cyclin D1)在香烟烟雾提取物(CSE)促进人肺动脉平滑肌细胞(HPASMCs)增殖中的作用和关系。方法用5%浓度的CSE干预同步化的HPASMCs。通过Western blot法检测PKCα蛋白在胞浆和胞膜中的表达。采用小干扰RNA (siRNA)技术分别剔除PKCα和cyclin D1基因表达。用实时荧光RT-PCR法检测cyclin D1 mRNA表达,Western blot法检测PKCα和cyclin D1蛋白的表达。采用四甲基偶氮唑盐比色法(MTT)和细胞计数法测定细胞增殖,流式细胞术观察细胞周期变化。结果5%CSE可激活PKCα,PKCα蛋白由胞浆转移至胞膜表达。通过Go6976抑制PKCα活性或采用siRNA技术剔除PKCα基因表达可显著抑制CSE介导的HPASMCs增殖和细胞周期的G1期向S期转换。5%CSE促进HPASMCs增殖中,cyclin D1mRNA和蛋白表达水平均明显升高。转染cyclin D1 siRNA可抑制CSE介导的HPASMCs增殖。此外,使用Go6976或转染PKCa siRNA可显著抑制CSE介导的HPASMCs中cyclin D1 mRNA和蛋白表达水平升高。结论香烟烟雾提取物通过蛋白激酶Cα途径上调周期蛋白D1表达促进人肺动脉平滑肌细胞增殖。更多还原

【Abstract】 Background and ObjectiveChronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, its prevalence rate is high, and so does the mortality it caused. Pulmonary vascular remodeling hinders the respiration process and may result in pulmonary hypertension, which is one of the principal factors on reducing the survival rate of COPD patients. Controlling pulmonary vascular remodeling can effectively prevent the formation of pulmonary hypertension, which is of great significance in the clinical treatment of COPD and cor pulmonale. Previous researchers mainly focused on the pathogenesis of hypoxic pulmonary hypertension and deem that hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodeling lead to the formation of pulmonary hypertension. In recent studies, it has been reported that pulmonary vascular remodeling occurs in smokers with normal lung functions and mild COPD patients without hypoxemia. Exposure to cigarette smoke for 6 months produces pulmonary hypertension in guinea pigs. These findings suggest that cigarette smoke may also promote pulmonary vascular remodeling, leading to pulmonary hypertension. The abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of major morphological features of pulmonary vascular remodeling induced by smoking. Cigarette smoke can result in an abnormal proliferation of pulmonary artery smooth muscle cells. However, the intrinsic mechanism has not been thoroughly understood yet.The proliferation of cells is closely related with cell cycle changes. While in a cell, cyclin plays a very important role in the regulation of cell cycle. In the Gl phase, cyclin D combines with cyclin-dependent kinase 4 (CDK4) or CDK6 to form complexes. These complexes phosphorylate retinoblastoma protein (Rb) and other family members. Phosphorylation of Rb activates the transcription factors and mediates the transcription of genes required for the cell to progress into S phase, which leads to the cell proliferation. Cyclin D1 is one of the major subtypes of cyclin D, and it regulates the G1/S phase transition. Cyclin D1 has been proven to be related to the proliferation of carcinoma cells. So far, few studies have been reported on the role of cyclin D1 in the vascular smooth-muscle cell proliferation. The effect of cyclin D1 on the proliferation of PASMCs remains to be understood.Protein kinase C (PKC), which is one of crucial members in serine-threonine kinase family, is a key intracellular signal transduction molecule. At present, PKC has been found with at least 12 isoforms, and functions of PKC isoforms in different cells vary greatly. Among the PKC isoforms, PKCαis generally reported to be a positive mediator for the vascular smooth muscle cells proliferation. Our previous study has shown that PKCαwas closely associated with the abnormal proliferation of the rat pulmonary arterial smooth muscle cells induced by cigarette smoking extract. However, the downstream mechanism of PKCαhas remained unclear so far. PKCαhas been implicated in the up-regulation of the cyclin D1 expression in several types of cultured cells. In HPASMCs, however, the existence of a similar mechanism needs to be proven.In this study, we investigate the pathobiology of human pulmonary artery smooth muscle cell proliferation induced by cigarette smoke from the perspective of the cell cycle control and the intracellular signal transduction. Our study may provide the basis for a new therapeutic approach for addressing COPD associated with pulmonary hypertension. Part one The changes of protein kinase Ca and cyclin D1 expressions in pulmonary artery from smokers with and without chronic obstructive pulmonary diseaseObjective The purpose of this study was to investigate the changes of protein kinase Ca (PKCa) and cyclin D1 expressions in pulmonary artery from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease (COPD). Methods The peripheral lung tissues were obtained from 10 nonsmokers with normal lung function (nonsmoker group),14 smokers with normal lung function (smoker group),11 smokers with mild to moderate COPD (COPD group). The morphological changes of pulmonary arteries were observed by hematoxylin-eosin staining. The expressions of a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), PKCa and cyclin D1 proteins in pulmonary artery smooth muscle (PASM) were determined by immunohistochemical staining. The percentages of PCNA-positive cells were considered as the smooth muscle cells proliferation index (PI). The mRNA expressions of PKCa and cyclin D1 in PASM were evaluated by real-time fluorescence PCR. Results Morphometry analysis showed that the ratio of pulmonary artery wall area to total area (WA%) in smoker group and COPD group was significantly greater than that in nonsmoker group (P< 0.01). The PASMCs proliferation index in smoker group and COPD group was obviously higher than that in nonsmoker group (P< 0.01). The protein levels of PKCa and cyclin D1 in PASMCs were markedly increased in smoker group and COPD group as compared with nonsmoker group (P< 0.01). The mRNA expressions of PKCa and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with nonsmoker group(P< 0.01). Significant correlations were found between PKCa protein and WA% or PI (P< 0.01). Correlations between cyclin D1 protein and WA% or PI also existed (P< 0.01). The expression of PKCa positively correlated with the expression of cyclin D1 at both protein and mRNA levels (P< 0.01). Conclusions Increased expressions of PKCa and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.Part two The role of cyclin D1 in human pulmonary artery smooth muscle cells proliferation induced by cigarette smoke extractObjective The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation induced by cigarette smoke extract (CSE). Methods Synchronized HPASMCs were treated with different concentrations of CSE (1%,2.5%,5%,10%,20%). The antisense eukaryotic expression vector of cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups:control group, vector group, antisense cyclin D1 group,5% CSE treated group, vector+5% CSE treated group and antisense cyclin Dl+5% CSE treated group. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. Results Low concentrations of CSE (1%,2.5%,5%,10%) stimulated proliferation of HPASMCs, with its maximal effect at concentration of 5%. On the contrary, high concentrations of CSE (20%) were the inhibitory of cell proliferation as a result of cytotoxicity. The antisense eukaryotic expression vector of cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P < 0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P< 0.05), and the indicators of cell proliferation were increased as well (P<0.05). The cyclin D1 mRNA and protein levels, the indicators of cell proliferation in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P< 0.05). Conclusions CSE could promote HPASMCs proliferation through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation stimulated by CSE.Part three Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase Cα-dependent induction of cyclin D1Objective Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) in vivo and in vitro. However, the molecular mechanism remains unclear. This study was aimed to investigate the role of signaling pathways involving protein kinase C alpha (PKCα) and cyclin D1 in the cigarette smoke extract (CSE)-induced HPASMCs proliferation. Methods Synchronized HPASMCs were treated with 5% CSE. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCαwas measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blot analysis. Small interference RNA (siRNA) was used to knockdown PKCa and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCa and cyclinDl protein levels were detected by Western blotting. Results CSE (5%) led to PKCa activation. Inhibition of PKCa activity by using Go 6976 or siRNA-mediated knockdown of PKCa significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by CSE (5%) at both mRNA and protein levels. CSE-stimulated cell proliferation and G1/S transition was markedly abolished by cyclin Dl siRNA. Moreover, Go 6976 or PKCa siRNA significantly suppressed CSE-induced upregulation of cyclin Dl at both mRNA and protein levels. Conclusions Cigarette smoke extract promotes HPASMCs proliferation through protein kinase C alpha-dependent induction of cyclin D1更多还原