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四种细胞来源生物标志物在急性呼吸窘迫综合征大鼠中的变化特点

The changes of four cell-resource biomarkers in rats with acute respiratory distress syndrome

摘要:

目的:观察脂多糖(lipopolysaccharide,LPS)诱导急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)大鼠的肺表面活性蛋白B(surfactant proteins B,SP-B)、纤溶酶原活化抑制因子(plasminogen activator inhibitor-1,PAI-1)、高迁移率族蛋白B1(high-mobility group box B1,HMGB1)和可溶性晚期糖基化终产物受体(soluble receptor for advanced glycation end products,sRAGE)等四项生物标志物的早期变化特点,为临床联合检测提供依据。方法:实验在中山大学心肺脑复苏研究所完成,36只雄性SD大鼠随机(随机数字法)分成LPS组和对照组,每组18只;LPS组采用肺内注射LPS 10 μg/mL诱导建立ARDS模型,对照组则肺内注射等量生理盐水,在造模后6 h、12 h、24 h留取血清、肺泡灌洗液及肺组织,每个时间点各6只大鼠,动脉血气及病理分析确认建模成功后用ELISA、免疫组化及Western blot检测SP-B、PAI-1、HMGB1和sRAGE的表达变化。采用成组 t检验和重复测量的方差分析比较两组间各指标的差异。 结果:LPS组PaO 2(mmHg)随时间进行性降低,且与对照组比较差异有统计学意义(80.38±1.74 vs 84.17±1.96;72.20±1.70 vs 81.26±1.57;68.52±2.45 vs 81.45±1.51, P<0.05),HE染色提示肺损伤程度持续恶化。LPS组血清中HMGB1(ng/mL) (0.34±0.08,0.43±0.12,0.50±0.03),SP-B(ng/mL)(2.21±0.11,2.74±0.15,3.07±0.28)和PAI-1(ng/mL)(40.38±3.06,50.02±4.82,57.34±4.67)进行性升高,与对照组比较差异统计学意义(均 P<0.05),但sRAGE(ng/mL)与对照组相比进行性下降(0.17±0.04,0.14±0.04,0.11±0.02),仅24 h与其他时点差异有统计学意义( P<0.05)。LPS组肺泡灌洗液中HMGB1(ng/mL)(0.08±0.02,0.13±0.02,0.21±0.03)、PAI-1(ng/mL)(48.88±4.17,56.66±4.01,65.83±4.96)和SP-B(ng/mL)(0.68±0.13,0.80±0.17,1.12±0.14)进行性升高;而sRAGE(ng/mL)进行性下降(0.14±0.05,0.12±0.02,0.11±0.02),且与对照组比较差异有统计学意义( P<0.05)。免疫组化分析提示6 h起PAI-1在肺间质逐步上调表达而HMGB1和SP-B则呈现肺泡内向肺实质扩散表达的趋势,但sRAGE则随时间呈下调表达趋势。Western blot提示SP-B、PAI-1和HMGB1表达量随时间升高,sRAGE表达量随时间逐渐降低。 结论:ARDS早期病程中,SP-B、PAI-1和HMGB1在血清、支气管肺泡灌洗液及肺组织中呈一致性变化,选择其一或组合检测均可协助临床早期识别ARDS。

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abstracts:

Objective:To provide experimental evidence for panel determination in clinical practice, we characterized the early changes of surfactant proteins B (SP-B), plasminogen activator inhibitor-1 (PAI-1), high-mobility group box B1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE) in acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS).Methods:In the Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat Sen University, 36 SD rats were randomly (random number) divided into the LPS group and control group. ARDS rat model in the LPS group was established by intrapulmonary injection of LPS (10 μg/mL) synchronously intrapulmonary injection of saline in the control group. Serum, bronchoalveolar lavage fluid (BALF) and lung tissues were collected at 6, 12 and 24 h after ( n=6 at each time point). After confirmed the success of ARDS model by arterial blood gas (PaO 2) and pathology, we determinated the expressions of SP-B, PAI-1, HMGB1 and sRAGE by enzyme linked immunoassay, immunohistochemistry and Western blot. Paired t test and repeated measurement ANOVA were used for comparison of these biomarkers between the two groups. Results:Progressively decreases of PaO 2 (80.38±1.74 vs 84.17±1.96; 72.20±1.70 vs 81.26±1.57; 68.52±2.45 vs 81.45±1.51) and synchronously worse deterioration at HE pathology were found in the LPS group than those in the control group ( P<0.05). Compared to the control group, a progressively significant increases in HMGB1 (ng/mL) (0.34±0.08, 0.43±0.12, 0.50±0.03), PAI-1 (ng/mL) (40.38±3.06, 50.02±4.82, 57.34±4.67) and SP-B (ng/mL) (2.21±0.11, 2.74±0.15, 3.07±0.28) but significant decreases at sRAGE (ng/mL) (0.17±0.04, 0.14±0.04, 0.11±0.02) were revealed in the serum of LPS group ( P<0.05). Another similarly significant increases in HMGB1 (ng/mL) (0.08±0.02, 0.13±0.02, 0.21±0.03), PAI-1 (ng/mL) (48.88±4.17, 56.66±4.01, 65.83±4.96) and SP-B (ng/mL) (0.68±0.13, 0.80±0.17, 1.12±0.14), but significant decreases at sRAGE (ng/mL) (0.14±0.05, 0.12±0.02, 0.11±0.02) in the BALF of the LPS group compared with those in control group ( P<0.05). Immunohistochemistry showed a consistent up-regulating started at 6 h appeared at PAI-1 (from lung interstitium to lung parenchyma), HMGB1 and SP-B (from lung parenchyma to lung interstitium) while a down-regulating at sRAGE in the LPS group. Western blot showed the elevated expression of SP-B, PAI-1 and HMGB1 but the descent expression of sRAGE following the treating time in the LPS group. Conclusions:In the early stage of ARDS, SP-B, PAI-1 and HMGB1 synchronously changes in serum, and BLAF and lung tissue had the similar tendency, of which can help clinician to early diagnose and identify ARDS by choosing one or combining the biomarkers.

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