Ⅱ型肺泡细胞Tfpi-1基因敲除对脂多糖诱导小鼠急性肺损伤的影响

目的 观察Ⅱ型肺泡细胞组织因子途径抑制物1(tissue factor pathway inhibitor 1,TFPI-1)基因敲除对细菌脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性肺损伤(acute lung injury,ALI)的影响。方法 将Tfpi-1flox/flox转基因小鼠和表面活性蛋白A (surfactant-associated protein A,Spa)-环化重组酶 (cyclization recombination enzyme,Cre)转基因小鼠交配, 获得Tfpi-1flox/flox:Spa-Cre基因型小鼠(C57BL/6J),使用免疫组化和荧光定量PCR检测不同基因型小鼠肺组织中TFPI-1的表达差异。LPS诱导ALI 24 h后运用体积描记法和有创呼吸功能检测法检测小鼠的呼吸功能;通过肺泡灌洗,计数肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中白细胞的数目；BCA法测定BALF中总蛋白水平；ELISA测定BALF中纤溶酶原激活物抑制因子1(plasminogen activator inhibitor 1,PAI-1)的浓度,HE染色观察肺损伤的程度;计算肺湿/干重比(wet/dry weight ratio,W/D)。结果 未干预情况下,Tfpi-1flox/flox:Spa-Cre基因型小鼠相对于野生型小鼠肺部结构和呼吸功能无明显变化。LPS诱导后,Tfpi-1flox/flox:Spa-Cre基因型小鼠相对于野生型小鼠肺损伤分数、BALF中白细胞总数、W/D和BALF中PAI-1浓度显著升高,潮气量(tidal volume,TV)、每分通气量(minute ventilation,MV)、吸气时间(inspiratory time,Ti)、肺动态顺应性(dynamic lung compliance,C)有降低趋势,而呼气时间(expiratory time,Te)、气道阻力(airway resistance,R)有升高趋势。结论 Ⅱ型肺泡细胞条件性敲除Tfpi-1基因促进了LPS诱导的小鼠ALI, 影响了小鼠的呼吸功能。
Objective To investigate the effect of conditional knockout of tissue factor pathway inhibitor 1 (TFPI-1) gene in type Ⅱ pneumocytes on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods Tfpi-1flox/floxtransgenic mice and surfatcant-associated protein A (Spa)-cyclization recombination enzyme (Cre) transgenic mice were inbreeded to produce Tfpi-1flox/flox:Spa-Cre mice.The expression of Tfpi-1 from the lung of mice was detected by immunohistochemistry and real-time PCR method. LPS was used to induce ALI. At 24 hours after LPS exposure, respiratory function was measured by plethysmography and invasive respiratory function test. Then the mice were euthanized to obtain bronchoalveolar lavage fluid (BALF), and the number of leukocyte were counted. The levels of protein concentration in BALF were measured by BCA methods, and the concentration of plasminogen activator inhibitor-1 (PAI-1) was measured by ELISA. Lung injury was observed by HE staining,and wet/dry weight ratio (W/D) were measured. Results Solo Tfpi-1 knockout in type Ⅱ pneumocytes did not affect the lung structure and respiratory function in Tfpi-1 flox/flox：Spa-Cre mice compared with wild-type mice.After induced by LPS, lung injury score, total number of leukocytes, W/D and the concentration of PAI-1 in BALF were significantly elevated. The respiratory function such as tidal volume (TV), minute ventilation (MV), inspiratory time (Ti),and dynamic lung compliance (C) were slightly decreased in Tfpi-1flox/flox:Spa-Cre mice,while expiratory time (Te) and airway resistance (R) were slightly increased compared with wild-type mice. Conclusions Conditional