超声在肺纤维化中的应用进展

肺纤维化是一类肺间质疾病,可分为原因不明的特发性肺纤维化和继发于其他原因的肺纤维化.小部分患者病情可长期稳定,大部分患者病情进展缓慢,个别患者病情进展迅速.因此早期诊断和及时干预对改善肺纤维化患者预后至关重要.随着超声技术的发展,其在肺部疾病诊断、疗效评估及病情进展监测中的应用越来越多.本文就超声在肺纤维化诊断、治疗随...     

博莱霉素致小鼠肺纤维化模型的动态演变及其发生机制

目的 了解小鼠肺纤维化模型的动态演变，探讨博莱霉素致肺纤维化的作用机制。方法 36只雄性ICR小鼠，按随机数字表法分为阴性对照组（NC组）和肺纤维化模型组（FMA、FMB、FMC、FMD、FME组），每组6只。除NC组外，其余各组经鼻滴入博莱霉素建立肺纤维化模型。分别于6、14、21、28和35d处死各组小鼠，取外周血经流式细胞仪测定T细胞亚群Th1／Th2和Tc1／Tc2，计数支气管肺泡灌洗液（BALF）中总细胞数和细胞分类，右肺行苏木素-伊红（HE）及Masson染色，测定左肺组织中羟脯氨酸（HYP）的含量；并提取左肺组织总RNA，采用半定量逆转录-聚合酶链反应（RT—PCR）了解肺组织中多种细胞因子的表达。测定35d处死小鼠的潮气量（Vt）、0．1秒用力呼气容积／用力肺活量（FEV0．1／FVC）和静态肺顺应性（Cst）。结果 ①肺纤维化各组小鼠BALF中细胞总数与NC组比较均显著增高（P均〈O．01），且其肺组织中HYP含量除FMA组外均显著升高（P均〈0．01）。②FME组VT和Cst均较NC组明显降低（P均〈0．01），FEV0．1／FVC则显著升高（P〈O．05）。③在肺纤维化模型炎症期（FMA组），T细胞Th1／Th2和Tc1／Tc2之间的平衡呈Th1、Tc1优势表达为主；在纤维化形成期（FMB、FMC组），则以Th2、Tc2优势表达；在肺纤维化形成后，Th1、Tc1再度呈优势表达的状态。④肺纤维化模型组中转化生长因子-β1（TGF—β1）和金属蛋白酶组织抑制因子-1（TIMP-1）均较NC组显著升高（P均〈0．01）。结论 博莱霉素致肺纤维化小鼠肺功能为限制性通气功能障碍，Th2、Tc2及促纤维化生成因子在肺纤维化形成过程中发挥重要作用。     

Biochemical and histological changes in pulmonary fibrosis induced in rabbits with intratracheal bleomycin

Pulmonary fibrosis was induced in rabbits by an intratracheal instillation of bleomycin. Histologically, at 2 weeks there was inflammation but only limited evidence of increased collagen deposition; at 8 weeks the inflammatory response had subsided and increased collagen deposition, characteristic of early interstitial fibrosis, was observed. Biochemical analyses showed bleomycin treatment caused marked increases in the total amounts of RNA, DNA, mixed protein, collagen and elastin when compared to controls (P less than 0.001 in all cases). Furthermore the increases were essentially complete by 2 weeks where the contents had increased by 110 +/- 13%, 60 +/- 11%, 148 +/- 12%, 94 +/- 15% and 89 +/- 11% respectively (P less than 0.001 in all cases). When collagen and elastin were expressed as concentrations with respect to wet weight, total protein or DNA content, the changes were not statistically significant. No changes were observed in the relative amounts of type I and type III collagen. It is concluded that: (1) compared to biochemical analysis, histology is relatively insensitive in detecting the early increases in connective tissue proteins; (2) measurements of lung collagen and elastin should be expressed as total lung contents wherever possible; the concentration of these proteins may remain unchanged, especially in the early stages of fibrosis, due to concomitant increases in other lung constituents; (3) changes in the relative amounts to types I and III collagens do not play a major role in the pathology of this form of pulmonary fibrosis.     

抗中性粒细胞胞浆抗体检测在肺纤维化疾病中应用研究

目的 探讨肺纤维化疾病患者的抗中性粒细胞胞浆抗体（ANCA）与血清胱抑素C（CysC）和高敏C反应蛋白等指标之间的变化规律.方法 检测69例肺间质纤维化伴感染患者和30例健康对照组的ANCA及血尿素氮、肌酐、CysC和高敏C反应蛋白等指标.ANCA检测先应用间接免疫荧光法,并用免疫印迹法确定靶抗原.生化指标用全自动生化分析仪测定.白细胞和血小板用血细胞分析仪测定.结果 肺纤ANCA（＋）组CysC水平明显高于肺纤ANCA（-）组与健康对照组（P〈0.05）,而肺纤ANCA（-）组与健康对照组的CysC比较差异无统计学意义（P〉0.05）;肺纤ANCA（＋）组与肺纤ANCA（-）组CRP水平明显高于与健康对照组（P〈0.01）,其他指标各组比较差异无统计学意义（P〉0.05）.结论 肺纤ANCA（＋）可能伴有肾功能损伤,应注重肺纤ANCA（＋）患者的肾功能监测.     

血浆蛋白C及蛋白S在肺栓塞患者血清中的表达及意义

目的：测定肺栓塞患者血浆蛋白C及蛋白S的表达,探讨其联合检测在临床中的意义。方法：肺栓塞患者132例为观察组,体检正常成人132例为正常组,均检测血清中血浆蛋白C及蛋白S的表达,分析其不同预后与血浆蛋白C及S的差异及二者间的相关性。结果：与正常组比较,观察组患者血清中血浆蛋白C及蛋白S均呈明显低表达,其中死亡病例更低于好转病例,好转病例明显低于治愈病例（P〈0.05,0.01）;线性相关分析,血清中血浆蛋白C与蛋白S的表达呈正相关。结论：血浆蛋白C及蛋白S在肺栓塞患者血清中均呈低表达,且与预后有关,早期检测血浆蛋白C及蛋白S可能对判断肺栓塞的病情及指导治疗有重要价值。     

ELISA法测定血清S100B蛋白

目的建立ELISA测定血清S100B蛋白的方法。方法制备S100B单克隆和多克隆抗体,以双抗体夹心法测定血清S100B蛋白。结果方法的平均回收率为93.7%-104%,批内及批间平均变异分别为3.7%和5.9%,方法的线性为0-12.5μg/l,参考值范围为0.14-0.38μg/l。结论该法简便、快速、特异、敏感,适于临床常规应用。     

Protein S declines during winter respiratory infections

Summary. There is an increase in cardiovascular and cerebrovascular morbidity and mortality in the older adult population during the winter that could be related to prothrombotic changes caused by seasonal effects or acute respiratory tract infections. Therefore, a prospective cohort study was conducted to assess the effect of acute winter respiratory infection on hemostatic parameters including complement 4b‐binding protein (C4‐BP), functional protein S, total protein S, free protein S, and the inflammatory marker, interleukin‐6 (IL‐6), in younger and older adults. The changes in the levels of hemostatic and inflammatory markers during winter respiratory infections in the younger and older adults were compared with matched, non‐infected controls. In younger and older adults (combined), total protein S increased from 83% [95% confidence interval (CI); 77–88] to 98% (95% CI; 91–106, P < 0.001) while free protein S decreased from 100% (95% CI; 95–105) to 70% (95% CI; 66–75, P < 0.001). There were no significant changes in C4‐BP (P = 0.622), functional protein S (P = 0.061) or IL‐6 (P = 0.651) from baseline. In a multivariate analysis, only total protein S and free protein S showed significant association with seasonal change after adjusting for the effect of infection. The estimated effect of season on total protein S was 15 ± 4%, P < 0.001 and on free protein S was ?27 ± 3%, P < 0.001. After adjusting for seasonal effect, only functional protein S showed a significant association with infection, with the estimated effect of ?17 ± 5%, P < 0.001. The results in the younger and older adults were similar to those in the combined groups. Seasonal and infection‐related changes in hemostatic parameters including an increase in fibrinogen and a decrease in free protein S, observed in this study, may contribute to thrombotic risk and excess vascular disease morbidity and mortality in older populations in the winter season.     

Efficacy and safety of recombinant human soluble thrombomodulin (ART-123) in disseminated intravascular coagulation: results of a phase III, randomized, double-blind clinical trial

BACKGROUND: Soluble thrombomodulin is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. OBJECTIVES: We conducted a multicenter, double-blind, randomized, parallel-group trial to compare the efficacy and safety of recombinant human soluble thrombomodulin (ART-123) to those of low-dose heparin for the treatment of disseminated intravascular coagulation (DIC) associated with hematologic malignancy or infection. METHODS: DIC patients (n = 234) were assigned to receive ART-123 (0.06 mg kg(-1) for 30 min, once daily) or heparin sodium (8 U kg(-1) h(-1) for 24 h) for 6 days, using a double-dummy method. The primary efficacy endpoint was DIC resolution rate. The secondary endpoints included clinical course of bleeding symptoms and mortality rate at 28 days. RESULTS: DIC was resolved in 66.1% of the ART-123 group, as compared with 49.9% of the heparin group [difference 16.2%; 95% confidence interval (CI) 3.3-29.1]. Patients in the ART-123 group also showed more marked improvement in clinical course of bleeding symptoms (P = 0.0271). The incidence of bleeding-related adverse events up to 7 days after the start of infusion was lower in the ART-123 group than in the heparin group (43.1% vs. 56.5%, P = 0.0487). CONCLUSIONS: When compared with heparin therapy, ART-123 therapy more significantly improves DIC and alleviates bleeding symptoms in DIC patients.     

Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fibrosis

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-beta(1) has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-beta(1) in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-beta(1)-induced responses have not been defined. To address these issues, we targeted bioactive TGF-beta(1) to the murine lung using a novel externally regulatable, triple transgenic system. TGF-beta(1) produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-beta(1)-induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-beta(1)-induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-beta(1) in vivo and define the critical role of Egr-1 in the TGF-beta(1) phenotype. They also demonstrate that Egr-1-mediated apoptosis is a prerequisite for TGF-beta(1)-induced fibrosis and remodeling.     

重组人血管生成素-1与肺间质纤维化血管正常化时相的关系

目的探讨重组人血管生成素1(Human Angiopoietin 1,rHuANG 1 )对小鼠模型肺间质纤维化微血管正常化的最佳时相及其分子机制。方法将40只C57/BL6小鼠按掷硬币法随机分成对照组和实验组,每组各20只。对照组腹腔注射生理盐水（NS）(0.2 ml/d);实验组腹腔注射rHuANG 1(5 mg·kg-1·d-1)。每组分别于治疗后的第2、4、6、9天处死5只小鼠留取标本。测量小鼠肺组织羟脯氨酸含量变化。酶联免疫吸附测定法（ELISA）检测肺组织中G蛋白调节信号5 (regulator of G protein signaling 5, RGS5)蛋白浓度,苏木精伊红(HE)染色检测肺组织病理变化,免疫组织化学形态学分析血管内皮生长因子(vascular endothelial growth factor, VEGF)的变化。结果①成功建立C57/BL6小鼠肺间质纤维化模型。②实验组接受rHuANG 1注射后,第6天,第9天肺羟脯氨酸含量降低（P<0.01）。③ ELISA结果显示,实验组肺组织中RGS5的表达在治疗第4天和第6天与对照组比较明显增高（P<0.05）。④HE染色结果显示,实验组和对照组不同时间点均可见不同程度的肺纤维化。对照组坏死程度高于实验组。其中,实验组第4天和第6天组织修复最明显,而对照组不同时点肺间质纤维化比较明显。⑤免疫组织化学结果表明,与对照组相比,实验组第4、6天VEGF在肺间质纤维化中的表达率升高明显减少（P<0.05）。结论rHuANG 1作用于小鼠肺间质纤维化后第4～6天可作为血管正常化时间窗,可能与VEGF相关。     

Protein S multimers and monomers each have direct anticoagulant activity

BACKGROUND AND OBJECTIVES: Plasma protein S (PS) is an essential anticoagulant that has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric purified PS has poor PS-direct and that a subpopulation of multimeric purified PS has high PS-direct and high affinity for phospholipids. We independently examined the relative PS-direct and affinity for phospholipids of monomeric and multimeric PS and we obtained contrasting results. METHODS AND RESULTS: Unpurified recombinant protein S (rPS) was monomeric and had PS-direct potency similar to that of both PS in plasma and multimeric affinity-purified PS, as measured in plasma assays for PS-direct and in thrombin-generation assays. Multimers of unpurified rPS were not induced by ethylenediaminetetraacetic acid (EDTA), pH 2.5, NaSCN, or barium adsorption/elution. Multimers were induced by chromatography in the presence of EDTA and thus may be concentration-dependent. In contrast to a different report, monomers, dimers, trimers, and higher-order PS forms were clearly separated in sedimentation velocity experiments and multimers were not dissociated by adding Ca(2+). Active plasma-derived and recombinant immunoaffinity-purified PS were fractionated into monomers and multimers. On a mass basis, monomers and multimers had similar specific PS-direct and ability to compete with prothrombinase components (factors Xa/Va) for limiting phospholipids. FXa ligand blotted to both monomers and multimers. CONCLUSIONS: Plasma PS-direct is similar to that of affinity-purified PS and unpurified rPS. Under our conditions, monomeric and multimeric PS have similar PS-direct and ability to compete for phospholipids. Discordant earlier findings are likely due to loss of PS-direct during conventional purification procedures.     

肺纤维化大鼠血清克拉拉细胞蛋白和表面活性蛋白-D动态变化及早期诊断意义

目的 探讨克拉拉细胞蛋白(CC16)和表面活性蛋白-D(SP-D)在肺纤维化大鼠中的表达水平及早期诊断意义.方法 Wistar大鼠60只随机分为正常对照组(对照组)、博莱霉素致肺纤维化组(模型组).每组30只,分别于造模后1、3、7、14、28 d处死,采用HE、Masson染色观察其肺组织病理变化,碱水解法测定肺组织羟脯氨酸含量,酶联免疫吸附法测定血清CC16、SP-D水平.结果 模型组大鼠肺组织羟脯氨酸含量自第7天[(913.1±69.3)μg/g]起,较对照组[(790.5±36.8)μg/g]升高(P<0.05);血清CC16自第3天[(27.34±0.32)μg/L]开始,低于对照组[(27.85 ±0.32)μg/L](P<0.05),并随病情进展而逐渐降低;血清SP-D各时相均高于对照组(P均<0.05),并且随病情进展而逐渐升高.结论 血清CC16、SP-D在大鼠肺纤维化早期有明显改变,其水平变化可能为肺纤维化的早期诊断提供生物学指标.     

Protein Z, protein S levels are lower in patients with thrombophilia and subsequent pregnancy complications

OBJECTIVE: We posit that low levels of protein S (PS) and protein Z (PZ) contribute to adverse pregnancy outcome (APO). PATIENTS: We evaluated 103 women with subsequent normal pregnancy outcome (NPO), 106 women with APO, and 20 women with thrombophilia (TP). METHODS: We compared first trimester (1st TRI) PZ levels in 103 women with NPO, 106 women with APO, and in 20 women with TP. We compared plasma levels of PZ and free PS antigen during the second (2nd TRI) and third trimesters (3rd TRI) of pregnancy in 51 women with APO and 51 matched women with NPO. RESULTS: The mean 1st TRI PZ level was significantly lower among patients with APO, compared to pregnant controls (1.81 +/- 0.7 vs. 2.21 +/- 0.8 microg mL(-1), respectively, P < 0.001). Of patients with known TP, those with APO had a tendency for lower mean PZ levels compared to those TP women with NPO (1.5 +/- 0.6 vs. 2.3 +/- 0.9 microg mL(-1), respectively, P < 0.0631). There was a significant decrease in the PZ levels in patients with APO compared to NPO (2nd TRI 1.5 +/- 0.4 vs. 2.0 +/- 0.5 microg mL(-1), P < 0.0001; and 3rd TRI 1.6 +/- 0.5 vs. 1.9 +/- 0.5 microg mL(-1), P < 0.0002). Protein S levels were significantly lower in the 2nd and 3rd TRIs among patients with APO compared to patients with NPO (2nd TRI 34.4 +/- 11.8% vs. 38.9 +/- 10.3%, P < 0.05, respectively; and 3rd TRI 27.5 +/- 8.4 vs. 31.2 +/- 7.4, P < 0.025, respectively). CONCLUSIONS: We posit that decreased PZ and PS levels are additional risk factors for APO.     

Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1.

Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.     

Direct effects of protein S in ameliorating acute lung injury

Summary. Objective: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. Methods: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. Results: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS‐treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor‐α, interleukin‐6 and monocyte chemoattractant protein‐1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor‐α and interleukin‐6 in the lung compared with untreated animals. Thrombin‐antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. Conclusion: Protein S protects against LPS‐induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.     

声脉冲辐射力弹性成像与生化S指数评定兔肝纤维化的实验研究

目的探讨声脉冲辐射力弹性成像(ARFI)技术与血清生化指标S指数在兔肝纤维化分期中的诊断价值。方法 32只家兔皮下注射5%硫代乙酰胺制备肝纤维化模型(实验组),8只家兔于颈背部皮下注射生理盐水(对照组)。于造模第4、8、12周末行ARFI和血清实验室检查,获取肝脏剪切波速度(SWV),计算S指数,取肝脏制作标本行病理肝纤维化分期,比较SWV与S指数在兔肝纤维化分期中的诊断价值。结果实验组中29只家兔造模成功,肝纤维化分期S1、S2、S3、S4期分别为10只、8只、7只、4只;另3只家兔肝脏严重炎症坏死而无纤维化。SWV与S指数均随着纤维化程度的加重而增大,各组间差异均有统计学意义(P﹤0.05);SWV较S指数与肝纤维化病理分期具有更显著的相关性(r=0.724、0.472,P﹤0.05);应用SWV和S指数诊断肝纤维化程度≥S1、≥S2、≥S3、S4对应的ROC曲线下面积分别为0.92、0.87、0.82、0.89和0.76、0.75、0.71、0.74。结论 ARFI较生化S指数能更准确地诊断兔肝纤维化程度。