急性肺损伤大鼠肺组织神经导向因子Slit2及Robo4的表达

Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury

目的观察神经导向因子Slit2及Rob04在肺组织的表达并探讨二者在ALI发病中的作用。方法在南华大学实验动物部将48只SD雄性大鼠随机（随机数字法）分为对照组（n=24）与ALI组（n=24）。ALI组采用盲肠结扎穿孔术复制急性肺损伤模型，对照组只开腹关腹，不行盲肠结扎穿孔术，以血清炎症因子浓度、动脉血氧分压（PaO2）、组织水肿、肺部特征性病理改变作为ALI模型成功主要指标。两组又分为术后12、24、48h3个组，每组8只。取大鼠动脉血测定PaO2；取肺组织测定湿／干质量（W／D）比值，并观察肺毛细血管通透性及组织病理学改变；ELISA检测血清TNF-α水平；RT-PCR测定肺组织Slit2及Rob04mRNA表达，免疫组织化学法观察蛋白表达情况。采用SPSSl3．0统计软件行单因素方差分析，P〈0．05为差异具有统计学意义。结果与对照组比较，Au组致伤后各时间点PaO2均降低，肺W／D比值、肺毛细血管通透性、血清TNF-α水平均升高（P〈0．05），病理观察显示肺组织受损；RT-PCR结果显示ALI组12h，24h，48h肺组织Slit2mRNA表达量较同期对照组下降[（0．56±0．13）V8．（0．87±0．05），F=41．39，P〈0．05，（O．42±0．10）vs．（0．85±0．07），F=93．54P〈0．05，（0．26±0．08）vs．（0．89±0．09），F=227．05，P〈0．05]；ALI组肺组织Robo4mRNA表达量与同期对照组比较，差异无统计学意义[（0．86±0．07）vs．（0．83±0．05），F=0．695，P〉0．05，（0．82±0．05）VS．（0．89±0．08），F=2．061，P〉0．05，（0．85±0．08）vs．（0．86±0．05），F=0．035，P〉0．05]；免疫组织化学研究显示Slit2主要表达于内皮细胞膜上，亦可表达于肺上皮细胞胞核及胞浆，Robo4则只表达于内皮细胞膜上；ALI组3个时间点肺组织Slit2蛋白表达量较同期对照组下降[（0．37±0．05）vs．（0．45±0．07），F=6．82，P〈0．05，（0．32±0．06）VS．（0．47±0．09），F=23．54，P〈0．05，（0．28±0．07）vs．（0．46±0．06），F=28．01，P〈0．05]；与对照组比较，ALI组肺组织Robo4蛋白表达量差异无统计学意义[（0．53±0.04）vs．（0．52±0.05），F=0．155，P〉0．05，（0．53±0．09）vs.（0．50±0．05），F=0．498，P〉0．05，（0．55±0．06）vs．（0．56±0．07），F=0．073，P〉0．05]。结论对照组大鼠肺组织可表达Slit2及Robo4，盲肠结扎穿孔致ALI大鼠肺组织Slit2表达下降，这可能与ALI发病有关。

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury （ALI） and explore the function of Slit2 and Robo4 in ALI. MethodsForty-eight Sprague-Dawley rats were randomly （random number） divided into control group （n = 24） and ALI group （n = 24）. ALI model was reproduced by cecum ligation and puncture （CLP）. The control group only experienced a simulated operation without CLP. Both groups were further divided into 3 subgroups with 8 rats in each subgroup： 12 h, 24 h, and 48 h subgroups, artery blood gas analysis, lung tissue wet/dry weight （W/D） ratio, lung histopathologic changes, pulmonary microvascular permeability were observed. The serum tumor nocrosis factor-α （TNF-α） was measured with enzyme linked immunosorbent assay （ELISA）. The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry. Date were analyzed by one-way ANOVA with SPSS version 13.0 software. Statistical significance was established at a P value of less than 0. 05. Results Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood （PaO2） decreased significantly, lung W/D weight ratio and pulmonary microvascular permeability, the serum TNF-α increased significantly （ all P 〈 0. 05 ）, histopathology of lung revealed signs of injury. The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [ （0.56±0.13）vs. （0.87±0.05）, F=41.39, P〈0.05, （0.42±0.10） vs. （0.85±0.07）, F= 93.54, P〈0.05, （0.26±0.08） vs. （0.89±0.09）, F=227.05, P〈0.05]. but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [ （0.86±0.07） vs. （0.83±0.05）, F=0.695, P〉0.05, （0.82±0.05） vs. （0.89±0.08）, F= 2. 061, P 〉 0.05, （0. 86 ± 0.08） vs. （0. 86 ± 0. 05）, F = 0. 035, P 〉 0. 05 ]. Immunohistoehemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells, while lung epithelial cell nuclei and endoehylema. Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells. Compared with the control group, expression of Slit2 protein in lung tissue in ALl group decreased markedly [ （0.37±0.05） vs. （0.45±0.07）, F=6.82, P〈0.05, （0.32±0.06） vs. （0.47±0.09）, F=23.54, P〈0.05, （0.28±0.07） vs. （0.46±0.06）, F= 28.01, P 〈 0. 05 ]. As good as RT-PCR, there were no significant difference in expression of Robo4 protein in lung tissue between two groups [ （0. 53 ±0.04） vs. （0. 52 ±0.05）, F =0. 155, P 〉0. 05, （0. 53 ± 0.09） vs. （0.50±0.05）, F=0.498, P〉0.05, （0.55±0.06） vs. （0.56±0.07）, F=0.073, P 〉 0. 05 ]. Conclusions Lung tissues of control group rats express Slit2 and Robo4. The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALl caused by CLP may be associated with the occurrence of ALl.