AP-1在血管内皮细胞PAI-1表达中的作用

目的 研究核转录因子激活蛋白-l(activator protein-l,AP-1)在肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、弱氧化修饰低密度脂蛋白(minimal modified low density lipoprotein,mmLDL)诱导血管内皮细胞PAI-l基因表达中的转录调控机制。方法 进行人脐静脉内皮细胞(human vascular endothelial cells,HUVECs)的培养和鉴定。采用基因重组技术,构建4个含人PAI-l基因不同长度5′上游序列的荧光素酶报告基因质粒,并结合瞬时转染分析实验,初步确定PAI-l基因上游具有调控作用的是位于-823到-553之间的AP-l元件。为进一步确证AP-l元件的调控作用,采用PCR法对该元件进行定点突变。结果 TNF-α能明显诱导构建质粒pGL3-PAl-l-823／ 90荧光素酶的表达,但对质粒pGL3-PAl-l-823／ 90的3个突变体诱导作用明显降低;mmLDL也能明显诱导质粒pGL3-PAI-1-823／ 90荧光素酶表达,但对质粒pGL3-PAl-l-823／ 90的3个突变体的诱导作用不明显。结论 在TNF-α、mmLDL作用下,内皮细胞转录表达PAI-l基因过程中,3个AP-l元件起着重要的作用。     

Rhein inhibits transforming growth factor β1 induced plasminogen activator inhibitor-1 in endothelial cells

Objectives To investigate the effect of rhein on endothelial plasminogen activator inhibitor-1 (PAI-1) mRNA expression and protein production induced by transforming growth factor β1 (TGFβ1), and to explore the mechanism of the protective action of rhein on endothelial cells. Methods A human umbilical endothelium derived cell line (ECV-304) from ATCC was used in this study. The PAI-1 mRNA expression and protein synthesis in the endothelial cells were detected by Northern blot and flow cytometry analysis, respectively. The activity of phospho-p44/p42 MAP kinase induced by TGFβ1 was determined by immunoprecipitation analysis and western blot. Results TGFβ1 rapidly increased PAI-1 mRNA expression in the endothelial cells, and this effect lasted at least 24 hours. The upregulation of PAI-1 mRNA expression induced by TGFβ1 in endothelial cells was inhibited by rhein in a dose-dependent manner. In addition, rhein inhibited endothelial PAI-1 protein production. Further study revealed that rhein had a significant inhibitory effect on the activity of phospho-p44/p42 MAP kinase induced by TGFβ1 in human endothelial cells. Conclusions Our results showed that rhein may have a protective effect on the endothelial dysfunction by inhibiting overexpression of PAI-1, indicating a way for the treatment of vascular diseases.     

辛伐他汀对人脐静脉内皮细胞血管内皮生长因子表达影响的初步研究

目的:观察辛伐他汀对人脐静脉内皮细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体表达的影响.方法:将人脐静脉内皮细胞分为辛伐他汀组、TNF-α组、辛伐他汀+TNF-α组、IL-1β组、辛伐他汀+IL-1β组和空白对照组.采用免疫细胞化学方法,观察TNF-α和IL-1β对细胞中VEGF及其受体表达的影响,并观察辛伐他汀的干预作用.结果:TNF-α组和IL-1β组的VEGF及其受体表达明显高于空白对照组(P＜0.05),辛伐他汀十TNF-α组和辛伐他汀+IL-1β组表达分别明显低于TNF-α组和IL-1β组(P＜0.05).结论:辛伐他汀可以抑制炎性刺激导致的人脐静脉内皮细胞VEGF及其受体的表达.     

Activity of Ginkgo biloba extract and quercetin on thrombomodulin expression and tissue-type plasminogen activator secretion by human umbilical vein endothelial cells

INTRODUCTION Ginkgo biloba extract (GBE) has been used as a ommonly prescribed drug in traditional Chinese edicine for several thousand years. GBE is still sed in clinics in Europe to alleviate symptoms ssociated with blood circulation disorder. It has be…     

血管紧张素-(1-7)对氧化低密度脂蛋白诱导人血管内皮细胞纤溶功能异常的调节

目的 探讨血管紧张素-(1-7)[Ang-(1-7)]对氧化低密度脂蛋白(ox-LDL)诱导血管内皮细胞分泌组织型纤溶酶原激活物(t-PA)及其抑制物-1(PAI-1)的影响。方法 用酶消化法收集并培养人脐静脉内皮细胞(HUVECs),分别以不同终浓度的Ang-(1-7)(10、100、1000nmol/L)和ox-LDL[25、50、100mg/L(蛋白质含量)]进行单独或联合刺激,并以A-779(终浓度为100nmol/L)进行干预,孵育24h。以ELISA方法检测培养基上清液中t-PA和PAI-1蛋白含量,RT-PCR法检测PAI-1和t-PAmRNA的表达。结果 ox-LDL使PAI-1的分泌量显著增加,t-PA分泌显著降低,PAI-1mRNA表达升高,t-PAmRNA表达降低(P<0.001~0.05),并呈剂量依赖性;Ang-(1-7)使PAI-1分泌量及PAI-1mRNA表达显著降低,亦呈剂量依赖性(P<0.01~0.05),而对t-PA分泌及t-PAmRNA表达无影响;在混合刺激组中,与ox-LDL组比较,Ang-(1-7)使PAI-1及PAI-1mRNA表达降低、t-PA及t-PAmRNA表达升高(P<0.01~0.05),但PAI-1及PAI-1mRNA表达仍高于对照组,t-PA及t-PAmRNA表达仍低于对照组(P<0.05)。加入A-779后,Ang-(1-7)的作用消失。结论 Ang-(1-7)对氧化低密度脂蛋白诱导的血管内皮细胞纤溶异常具有显著的调节作用,此作用是通过特异性受体介导的。     

金属硫蛋白和同型半胱氨酸对人脐静脉内皮细胞PAI-1活性及mRNA表达的影响

目的探讨同型半胱氨酸(homocysteine,Hcy)与金属硫蛋白(metallothionein,MT)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)纤溶酶原激活物抑制剂(Plasminogen activator inhibitor-1,PAI-1)活性及mRNA表达的影响.方法0.1 mmol/L、0.5 mmol/L、1.0 mmol/L Hcy及1.0 mmol/L Hcy加不同浓度MT(1nmol/L,5 nmol/L,10 nmol/L,50 nmol/L)分别作用HUVECs 6 h后,用PAI-1发色底物法检测PAI-1活性;用RT-PCR技术检测PAI-1 mRNA.结果Hcy呈浓度依赖性地增加HUVECs PAI-1活性及其mRNA表达(P均＜0.01);不同浓度的MT均可显著降低Hcy所致的PAI-1活性及其mRNA表达的增高(P均＜0.05),且呈剂量依赖性.结论MT可降低Hcy所致的PAI-1活性及其mRNA表达增高作用.     

PDGF对大鼠肾小球系膜细胞表达PAI-1的影响

目的:探讨血小板源性生长因子(PDGF-BB)对体外培养的大鼠肾小球系膜细胞表达纤溶酶原激活物抑制剂-1(PAI-1)的影响.方法:以0、5、10 ng/ml终质量浓度的PDGF-BB刺激体外培养的大鼠肾小球系膜细胞分别作用0、12、24、48 h, 以发色底物显色法检测细胞上清液中PAI-1的活性;以0、5、10 ng/ml质量浓度的PDGF-BB刺激系膜细胞24 h和10 ng/ml质量浓度的PDGF-BB刺激细胞0、12、24、48 h, 半定量RT-PCR观察系膜细胞PAI-1 mRNA表达情况.结果:0、5、10 ng/ml的PDGF-BB 作用于系膜细胞24 h,其PAI-1 mRNA/GAPDH mRNA分别为0.296、 0.428、0.542.10 ng/ml的PDGF-BB作用系膜细胞0、12、24、48 h, PAI-1 mRNA/GAPDH mRNA分别为0.156、0.345、0.493、0.597,表现为浓度和时间依赖效应.PDGF-BB也可浓度依赖性地增加PAI-1的活性,24 h内活性逐渐增高,48 h时下降.结论:PDGF-BB可上调体外培养的大鼠肾小球系膜细胞PAI-1的活性及其mRNA的表达,提示PDGF可能通过抑制细胞外基质的降解导致细胞外基质的积聚,从而参与肾小球硬化的过程.     

激活蛋白-1对香烟烟雾提取物诱导气道 黏蛋白5AC基因表达的调控作用

目的：了解激活蛋白-1（AP-1）参与调控香烟烟雾诱导的气道黏蛋白（MUC）5AC表达的作用机制,探讨此过程中AP-1活化的可能信号途径。 方法：体外培养人气道上皮细胞BEAS-2B,给予香烟烟雾提取物（CSE）刺激,干预实验用c-Jun 的显性负性突变体TAM67转染细胞阻断AP-1的DNA结合活性;用SP600125和PD98059预处理分别阻断c-Jun氨基端激酶（JNK）和细胞外信号调节激酶（ERK）的活性。以ELISA法检测MUC5AC蛋白含量,Western印迹检测磷酸化JNK（p-JNK）、磷酸化ERK（ p-ERK）和磷酸化P38（ p-P38）含量,RT-PCR检测MUC5AC mRNA 表达水平, EMSA测定AP-1的 DNA结合活性。 结果：CSE（1 g/L）刺激后MUC5AC mRNA表达水平和蛋白含量明显高于对照组（均P＜0.01）,AP-1 的DNA结合活性也明显强于对照组（P＜0.01）,伴随p-ERK和p-JNK蛋白含量显著增加,与对照组相比,差异有统计学意义（均P＜0.01）,而P38含量无明显变化(P＞0.05);与CSE组相比,TAM67转染细胞后MUC5AC mRNA表达水平和蛋白含量均显著降低 (P＜0.01);与CSE组相比,用SP600125或PD98059处理细胞后可明显抑制AP-1的 DNA结合活性 (均P＜0.05),并下调MUC5AC蛋白含量和mRNA的表达（均P＜0.05）。 结论：AP-1参与调控CSE诱导的气道MUC5AC基因表达的过程,此过程是由JNK和ERK信号转导通路激活AP-1,再由AP-1与MUC5AC启动子上AP-1 DNA反应元件结合后在转录水平上调MUC5AC的表达而实现的。     

IL-6诱导血管内皮细胞纤溶相关蛋白的表达

目的:观察IL-6对脐静脉血管内皮细胞(HUVECs)表达组织纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物1(PAI-1)的影响.方法:用生长良好的第2、3代HUVECs细胞进行实验,用CCK-8测定IL-6(0.125、0.25、0.5、1.2 ng/ml)作用前后细胞活性;用发色底物法测定0.5 ng/ml IL-6组和对照组(不加IL-6)培养上清液中tPA、PAI-1活性;进一步用RT-PCR检测细胞内tPA和PAI-1 mRNA水平.结果:与对照组相比, IL-6(≤0.5 ng/ml)对细胞增殖活性的影响无差异性.0.5 ng/ml IL-6组tPA活性在12～72 h显著升高(P<0.05),且显著上调tPA mRNA,12 h达到峰值,以后渐降,48 h mRNA达正常水平.而IL-6组PAI-1活性与PAI-1 mRNA表达与对照组无差异性.结论: IL-6可活化内皮细胞,显著上调HUVECs的tPA mRNA的转录和tPA分泌,诱发纤溶系统活化,而对PAI-1 mRNA或PAI-1活性无影响.IL-6的这种效应在炎症反应的病理生理过程中可能发挥重要作用.     

内皮细胞微粒对人脐静脉内皮细胞VCAM-1和ICAM-1表达的影响

目的:探讨内皮细胞微粒(Endothelial microparticles,EMPs)对人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)血管细胞粘附分子-1(Vascular cellular adhesion molecule-1,VCAM-1)和细胞间粘附分子-1 (Intercellular adhesion molecule 1,ICAM-1)表达的影响.方法:取生长良好的第4、5代HUVECs,将细胞以1×105个/ml密度接种于5 cm2的培养皿中,待细胞生长近80％融合时进行干预.按0、1×102、1×103、1×104、1×105个ml浓度的EMPs进行分组及刺激,每组12个样本.在共同培养24h后收集细胞.分别采用实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测VCAM-1和ICAM-1 mRNA和蛋白的表达.结果:HUVECs受EMPs刺激,VCAM-1和ICAM-1 mRNA及蛋白表达显著增加,且EMPs的这种作用呈浓度依赖性增强(均P＜0.05).结论:EMPs能上调HUVECs VCAM-1和ICAM-1的表达,EMPs不仅是内皮功能障碍的标记物,还能加重内皮功能损害.     

同型半胱氨酸诱导人脐静脉内皮细胞MCPIP表达

目的 探讨同型半胱氨酸是否可以诱导人脐静脉内皮细胞中MCPIP的表达.方法 体外培养并鉴定人脐静脉内皮细胞;分别用10、50、100、200、500 μmol/L Hcy与HUVECs孵育24 h;用100 μmol/L Hcy与HUVECs分别孵育1、2、4、8、12、24 h;Real-time PCR检测MCPIP mRNA的表达,Western blot检测MCPIP蛋白质的表达.结果 Real-time PCR和Western blot分析结果表明,在所设置浓度下,Hcy均可诱导HUVECs中MCPIP的表达（P＜0.05）,其中以100 μmol/L Hcy作用24 h时MCPIP表达量最多;100 μmol/l Hcy与细胞作用2h时后MCPIP表达与对照组相比即有明显增高（P＜0.05）,并存在时间依赖效应.结论 Hcy可诱导HUVECs中MCPIP的表达,这可能与它致As的机理有关.     

Inhibitory Effects of Fenofibrate on Plasminogen Activator Inhibitor-1 Expression in Human Endothelial Cells

Endothelial cells ( ECs) are an i mportantsource of plasminogen activator inhibitor type-1(PAI-1) . PAI-1 is the major physiological inhibi-tor of fibrinolysis and has been considered as inde-pendent risk factor of coronary artery disease(CAD)[1].The higher content and activity of PAI-1 may reduce fibrinolysis ,then induce the i mbal-ance between coagulation and fibrinolysis . Fibrindepositing on vascular endothelial cells can not bedissolved easily , which promote thrombosis . Per-oxiso…     

hVEGF121基因转染对人脐静脉内皮细胞生长的作用

目的:构建人血管内皮生长因子(hVEGF)-121的真核细胞复制表达质粒,将hVEGF121基因转染进人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),通过旁分泌作用来加快其生长速度.方法:采用RT-PCR方法,从人胚胎成纤维细胞中克隆hVEGF121前体蛋白全长cDNA,构建重组真核表达质粒pcDNA3-hVEGF121,用FuGENE 6介导转染入HUVEC.收集转染的HUVEC培养上清,用ELISA法定量检测hVEGF蛋白的表达情况;绘制细胞生长曲线,检测hVEGF121促进HUVEC增殖的生物学活性.设转染pcDNA3空质粒载体和不转染任何DNA的HUVEC做对照.结果:(1)成功构建了pcDNA3-hVEGF121真核表达质粒,经测序证明基因序列与GenBank中核酸序列一致,且插入方向正确;(2)转染细胞瞬时表达hVEGF蛋白,于转染1d后每104个细胞每天可分泌hVEGF蛋白59.95 Pg,约为对照组的100倍;之后表达水平先快后慢下降,于转染7d后仍较对照组高近1倍;(3)转染细胞生长速度明显加快,在转染3 d后细胞数与对照组相比即有显著性差异(P＜0.01),细胞倍增时间从对照组的7d以上缩短为3d左右.结论:成功构建了pcDNA3-hVEGF121真核表达质粒;该质粒能在HUVEC中表达有生物活性的hVEGF121蛋白,通过旁分泌作用明显促进HUVEC的分裂增殖.     

组织型纤溶酶原激活剂在人胚胎表皮角质形成细胞分化中的作用

目的 探讨组织型纤溶酶原激活剂（tPA)参与人表皮角质形成细胞（KC）分化调控的作用。方法 采用免疫细胞化学（ICC）及原位杂交（ISH）技术定性、定量检测早、中、晚期人胚胎表皮KC中tPA蛋白及mRNA的表达。结果 （1）tPA在人胚胎期表皮KC中表达量明显高于出生后期。胚胎期的表达高峰在胚胎中期。胚胎晚期其蛋白水平开始降低。而tPAmRNA表达则维持在相对较高水平。（2）tPA在人胚胎期主要存在于分化程度高的表皮浅层KC内。（3）tPA聚集于KC胞膜下方。结论 tPA参与表皮KC分化的调控。     

2型糖尿病小鼠肾脏纤溶酶原激活物抑制物-1表达

目的探讨2型糖尿病动物模型KKAy小鼠肾脏纤溶酶原激活物抑制物-1(PAI-1)和组织纤溶酶原激活物(tPA)mRNA表达与肾脏细胞外基质蓄积的关系.方法选取8周龄雄性KKAy小鼠和C57BL-J小鼠,分别于16、20和24周处死.采用发色底物法检测不同周龄的KKAy小鼠及参照组C57BL/J小鼠的血浆PAI-1活性,RT-PCR法检测小鼠肾皮质tPA mRNA表达,原位杂交法检测肾皮质PAI-1 mRNA水平,免疫组织化学和免疫印迹法检测肾皮质层粘连蛋白表达.结果不同周龄KKAy小鼠肾皮质层粘连蛋白表达均高于同龄对照组(P＜0.05).2型糖尿病KKAy小鼠与C57BL/J小鼠血浆PAI-1活性的差异没有显著性(P＞0.05);但肾皮质tPA mRNA水平却显著降低,以16周时最为显著,仅为C57BL/J小鼠的47%,随周龄的增加,其差异逐渐减小.原位杂交显示KKAy小鼠PAI-1mRNA可在肾脏多部位表达,并以近端小管上皮细胞内的表达为主,且明显高于同龄C57BL小鼠.结论2型糖尿病KKAy小鼠早期肾病时,细胞外基质蛋白的蓄积可能和tPA mRNA表达下调及PAI-1 mRNA表达上调所致的纤溶酶降解细胞外基质作用降低有关.     

Activation of peroxisome proliferator-activated receptor α in human endothelial cells increases plasminogen activator inhibitor type-1 expression

Objective To investigate the effect of peroxisome proliferator-activated receptors(PPARs) activators on plasminogen activator inhibitor 1(PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism.Methods Human umbilical vein endothelial cells(HUVECs) were obtained from normal fetus,and cultured conventionally.Then the HUVEC were exposed to fatty acids and prostaglandin J2 in varying concentrations with fresh media.RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs.Transient co-transfection of PAI-1 promoter and PPARα gene or PPARγ gene to ECV304 was performed.Results PPARα,PPARγ and PPARγ mNRA in HUVECs were detected by RT-PCR.Treatment of HUVECs with PPARα and PPARγ activators-linolenic acid,linoleic acid,oleic acid and prostaglandin J2,but not with stearic acid could augment PAI-1 mRNA expression and protein secretion in a concentrationdependent manner.Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARαDNA in HUVECs throgh a transient gene transfection assay,although the mRNA expression of the 3 subtypes of PPAR with their activators were not changes compared with controls.Conclusions HUVECs express PPARs,PPARs activators may increase PAI-1 expression in endothelial cells(EC).Although PPARs expresslon was not enhanced after being stimulated by their activators in EC,the functionally active PPARαis probably involved in regulating PAI-1 expression in EC.     

Role of connective growth factor in plasminogen activator inhibitor-1 and fibronectin expression induced by transforming growth factor β1 in renal tubular cells

Background Connective tissue growth factor (CTGF) contributes greatly to renal tubulointerstitial fibrosis, which is the final event leading to end-stage renal failure. This study was designed to investigate the effects of CTGF antisense oligodeoxynucleotides (ODNs) on the expressions of plasminogen activator inhibitor-1 (PAI-1) and fibronectin in renal tubular cells induced by transforming growth factor β1 (TGF-β) in addition to the role of CTGF in the accumulation and degradation of renal extracellular matrix (ECM). Methods A human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipidmediated CTGF antisense ODNs were transfected into HKC cells. After HKC cells were stimulated with TGF-β1 (5 μg/L), the mRNA levels of PAI-1 and fibronectin were measured by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 and fibronectin in the medium were determined by Western blot and ELISA, respectively. Results TGF-β was found to induce tubular CTGF, PAI-1, and fibronectin mRNA expression. PAI-1 and fibronectin mRNA expression induced by TGF-β was significantly inhibited by CTGF antisenes. ODNs CTGF antisense ODNs also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 and fibronectin protein secreted into the medium. Conclusions CTGF may play a crucial role in the accumulation and degradation of excessive ECM during tubulointerstitial fibrosis, and transfecting CTGF antisense ODNs may be an effective way to prevent renal fibrosis.     

人参皂甙Rg1对同型半胱氨酸诱导人脐静脉内皮细胞凋亡的逆转作用

目的人参皂甙Rg1对同型半胱氨酸(homocy steine,Hcy)诱导人脐静脉内皮细胞(HUVECs)凋亡的逆转作用。方法体外培养HUVECs,采用台盼蓝染色方法筛选人参皂甙Rg1最适浓度。将细胞分为对照组(无任何诱导物)、Hcy组(10μmol·L-1Hcy)、Hcy+Rg1组(10μmol·L-1Hcy和40mg·L-1Rg1)和Rg1组(单独用40mg·L-1Rg1处理的细胞),作用时间为24h。琼脂糖凝胶电泳和末端脱氧核糖核苷酸转移酶介导的缺口末端标记法(TUNEL)染色检测细胞凋亡情况。结果台盼蓝染色结果显示:人参皂甙Rg1的最适浓度40mg·L-1。琼脂糖凝胶电泳结果显示:对照组和Rg1组未见凋亡条带,Hcy组可见清楚的细胞凋亡特征性的"梯状"条带,而Hcy+Rg1组可见不明显的细胞凋亡特征性的"梯状"DNA断裂条带。TUNEL染色结果显示:与对照组和Rg1组相比,Hcy组细胞凋亡数量显著增加(P<0.01);与Hcy组相比,Hcy+Rg1组细胞凋亡数量显著减少(P<0.01),凋亡百分比由33.733%下降至9.200%,但仍高于对照组和Rg1组(P<0.01)。结论 40mg·L-1人参皂甙Rg1可一定程度逆转Hcy诱导的HUVECs凋亡。     

尼古丁对人脐静脉内皮细胞凋亡及坏死的影响

目的不同浓度尼古丁对人脐静脉内皮细胞(HUVECs)凋亡及坏死的影响。方法用CD34以免疫组织化学法鉴定HUVECs。通过3种不同浓度的尼古丁(3、30、300 ng/ml)刺激HUVECs 24 h后,用流式细胞仪检测其凋亡及坏死率。结果低浓度尼古丁促进细胞凋亡最强,而随着尼古丁浓度的增加,细胞凋亡率反而逐渐降低,各组差异具有统计学意义(P<0.05);此外,随着尼古丁浓度增加,细胞坏死率也日益增多,各组差异具有统计学意义(P<0.05)。细胞坏死率与尼古丁浓度呈正相关(r=0.675)。结论尼古丁对HUVECs凋亡的影响具有浓度依赖性,细胞坏死率与尼古丁浓度呈正相关。     

纤溶酶原激活物抑制剂-1与糖尿病足发病关系的研究

目的: 探讨纤溶酶原激活物抑制剂-1(PAI-1)与糖尿病足(DF)发病的关系,为DF的防治提供依据。方法: 42例DF患者为DF组,38例糖尿病(DM)无DF患者为DMNDF组,45名体检健康者为对照组,比较3组间PAI-1、糖基化血红蛋白(HbA1c)、C反应蛋白(CRP)等指标水平的变化。结果: 对照组、DMNDF组、DF组PAI-1、HbA1c、CRP依次升高,且差异均有统计学意义(P < 0.05~P < 0.01)。结论: DF患者PAI-1水平较高;对2型DM患者进行积极干预治疗,降低PAI-1水平,提高纤溶活性,可能延缓动脉硬化、狭窄,减少DF的发生。