博莱霉素致肺纤维化大鼠凝血活性的变化及意义

目的了解肺纤维化大鼠支气管肺泡灌洗液(BALF)中凝血活性的改变与肺纤维化发病的关系。方法48只SD大鼠按完全随机设计方法分为2组,每组各24只。博莱霉素(BLM)组以博莱霉素A5(BLMA5,5mg/kg)气管内滴入建立肺纤维化模型,于造模后第7、14、28、40天测定BALF中标准血浆、乏因子Ⅶ血浆、乏因子Ⅹ血浆的复钙时间以了解促凝活性(PCA),并测定BALF中凝血酶的活性和转化生长因子β1(TGFβ1)的蛋白含量。对照组气管内注入生理盐水。结果BLM组BALF中标准血浆的复钙时间各时间点分别为(56±10)、(78±4)、(172±11)、(180±6)s,对照组分别为(190±10)、(186±8)、(184±6)、(185±6)s;BLM组BALF中凝血酶的活性各时间点分别为(1.26±0.03)、(0.82±0.05)、(0.28±0.03)、(0.28±0.02)μg/ml,对照组分别为(0.31±0.02)、(0.32±0.03)、(0.31±0.04)、(0.29±0.05)μg/ml;BLM组BALF中TGFβ1的含量各时间点分别为(310±36)、(220±30)、(109±12)、(96±11)ng/ml,对照组分别为(92±20)、(94±12)、(92±10)、(90±9)ng/ml,3个指标第7天和第14天与对照组比较差异均有统计学意义(P<0.01),第28天及第40天与对照组比较差异无统计学意义(P>0.01)。在BLM组,第7天和第14天,乏因子Ⅶ血浆的复钙时间分别为(123±12)、(162±4)s,乏因子Ⅹ血浆的复钙时间分别为(357±22)、(387±12)s,乏因子Ⅹ血浆的复钙时间大于乏因子Ⅶ血浆并大于标准血浆的复钙时间。在14d内,TGFβ1的含量与促凝活性和凝血酶的活性呈正相关。结论在肺泡炎期,BALF中的促凝活性和凝血酶的活性升高,这种高凝状态是由于活化的凝血因子Ⅶa激活凝血因子Ⅹ,启动外源性凝血途径所致;在肺纤维化期,它们的活性不升高。凝血因子、凝血酶可能通过促进TGFβ1的合成直接促进肺纤维化的发生和发展。

The change and significance of coagulation activity in bleomycin-induced lung fibrosis in rats

OBJECTIVE: To investigate the role of coagulation activity of bronchoalveolar lavage fluid (BALF) in the pathogenesis of lung fibrosis. METHODS: Fourty-eight Sprague-Dawley rats were randomly divided into 2 groups, 24 rats in each group. In the bleomycin (BLM) group, the lung fibrosis model was made by tracheal instillation of bleomycin A(5) (BLMA(5), 5 mg/kg). At day 7, 14, 28 and 40, the recalcification time of normal pooled plasma, factor VII and X deficiency plasma were measured for procoagulation activity (PCA), and the thrombin activity and the protein level of transforming growth factor beta(1) (TGF-beta(1)) in BALF were also measured. In the control group, normal solution was instillated into the lungs. RESULTS: In the BLM group, the recalcification time of normal pooled plasma in BALF at the four time points were (56 +/- 10), (78 +/- 4), (172 +/- 11) and (180 +/- 6) s respectively, while in the control group, were (190 +/- 10), (186 +/- 8), (184 +/- 6) and (185 +/- 6) s respectively. The thrombin activity at the four time points were (1.26 +/- 0.03), (0.82 +/- 0.05), (0.28 +/- 0.03) and (0.28 +/- 0.02) microg/ml respectively in the BLM group, but were (0.31 +/- 0.02), (0.32 +/- 0.03), (0.31 +/- 0.04) and (0.29 +/- 0.05) microg/ml respectively in the control group. The level of TGF-beta(1) at the four time points were (310 +/- 36), (220 +/- 30), (109 +/- 12) and (96 +/- 11) ng/ml respectively in the BLM group, but were (92 +/- 20), (94 +/- 12), (92 +/- 10) and (90 +/- 9) ng/ml respectively in the control group. The above measurements were significantly different in day 7 and 14 between the BLM group and the control group (P < 0.01), while the differences were not significant at day 28 and 40 (P > 0.01). In the BLM group, at day 7 and 14, the recalcification time of factor VII deficiency plasma was (123 +/- 12) and (162 +/- 4) s respectively; the recalcification time of factor X deficiency plasma was (357 +/- 22) and (387 +/- 12) s respectively; the recalcification time of factor X deficiency plasma was longer than that of factor VII deficiency plasma and that of normal pooled plasma. Within 14 day, the level of TGF-beta(1) was positively correlated with PCA and thrombin activity. CONCLUSIONS: During the period of alveolitis, the PCA and thrombin activity were upregulated in BALF, which was caused by activated factor VII activating factor X and the switching to the exogenous coagulation pathway. But during the period of lung fibrosis, their activities were not upregulated. These results suggest that the coagulation factor and thrombin might contribute to the development of pulmonary fibrosis by promoting production of TGF-beta(1).