探讨Notch信号通路对烧伤大鼠血清诱导的肺血管内皮细胞(PMVEC)细胞间黏附分子(ICAM)-1的影响。

42只SD大鼠(6～8周龄)中，随机选取30只，按随机数字表法分为假伤组(n＝15)和烧伤组(n＝15)，烧伤组大鼠于95 ℃热水浸浴背部18 s造成30%总体表面积Ⅲ度烧伤，假伤组大鼠于37 ℃水浴中浸浴背部18 s模拟致伤。伤后6、12、24、48、72 h，烧伤组随机分别取3只大鼠，腹主动脉采血，酶联免疫吸附试验(ELISA)法检测血清中ICAM-1含量，假伤组大鼠行相同检测。取剩下的12只大鼠(6～8周龄)中的6只，按前述方法造成30%总体表面积Ⅲ度烧伤，伤后24 h制备烧伤大鼠血清；剩下的6只大鼠不作处理，制备健康大鼠血清。切取10只出生3 d的SD大鼠的肺外边缘组织，组织块法培养大鼠PMVEC，倒置相差显微镜下观察原代细胞培养2、6 d后的形态特征，流式细胞术对原代培养7 d的细胞进行细胞鉴定。取处于对数生长期的第4代PMVEC进行实验，将细胞接种于6孔板中，待细胞生长至80%融合时按随机数字表法分为3组(每组设3个复孔)：对照组(培养液中分别加入体积分数10%健康大鼠血清)，二甲基亚砜(DMSO)+烧伤血清组、γ-分泌酶抑制剂(GSI)+烧伤血清组(分别加入3 μL/mL DMSO、75 μmol/L GSI，培养24 h后，加入体积分数10%烧伤大鼠血清刺激培养24 h)，ELISA法检测PMVEC上清中ICAM-1的含量；流式细胞仪检测各组PMVEC中ICAM-1的含量；采用蛋白质印迹法检测ICAM-1蛋白表达水平，计算相对蛋白表达量；检测PMVEC的细胞黏附能力。数据比较采用单因素方差分析和LSD-t检验。

(1)烧伤组大鼠伤后6、12、24、48、72 h血清中的ICAM-1含量分别为(19.77±3.03)、(22.09±3.65)、(22.44±4.04)、(25.40±2.51)、(26.37±3.07) pg/mL，显著高于假伤组的[(10.60±1.51)、(11.03±1.95)、(10.87±0.89)、(9.30±0.89)、(10.93±1.22) pg/mL]，2组比较差异均有统计学意义(t＝4.699、4.466、3.181、10.490、8.097，P<0.05)。(2)原代细胞培养2 d后可见细胞从组织块边缘移出，生长较慢；培养6 d后可见细胞融合，细胞呈铺路石样生长。对原代培养7 d的细胞进行流式细胞术鉴定，结果显示CD31阳性细胞占98.6%，确定为PMVEC。(3)血清刺激培养24 h后，对照组，DMSO+烧伤血清组、GSI+烧伤血清组细胞上清中ICAM-1含量分别为1.21±0.25、2.16±0.12、3.07 ±0.30，3组比较差异有统计学意义(F＝46.72，P<0.05)；与对照组比较，DMSO+烧伤血清组和GSI+烧伤血清组细胞上清中ICAM-1含量均明显增加，差异均有统计学意义(t＝5.977、8.274，P<0.05)；GSI+烧伤血清组细胞上清中ICAM-1含量明显高于DMSO+烧伤血清组，2组比较差异有统计学意义(t＝4.847，P<0.05)。(4)血清刺激培养24 h后，对照组，DMSO+烧伤血清组、GSI+烧伤血清组PMVEC中ICAM-1平均荧光强度分别为2 582±143、3 453±204、4 559±414，3组比较差异有统计学意义(F＝37.84，P<0.05)；与对照组比较，DMSO+烧伤血清组、GSI+烧伤血清组细胞中ICAM-1含量均明显增加，差异均有统计学意义(t＝6.056、7.817，P<0.05)；GSI+烧伤血清组ICAM-1含量明显高于DMSO+烧伤血清组，2组比较差异有统计学意义(t＝4.149，P<0.05)。(5)血清刺激培养24 h后，对照组、DMSO+烧伤血清组、GSI+烧伤血清组的ICAM-1/GAPDH比值分别为0.74±0.08、1.07±0.08、1.38±0.28，3组总体比较差异有统计学意义(F＝30.76，P<0.05); DMSO+烧伤血清组和GSI+烧伤血清组的ICAM-1/GAPDH比值均明显高于对照组，差异均有统计学意义(t＝5.182、6.990，P<0.05)；GSI+烧伤血清组ICAM-1/GAPDH比值明显高于DMSO+烧伤血清组，差异有统计学意义(t＝3.770，P<0.05)。(6)PMVEC与人单核细胞系THP-1细胞共培养2 h后，倒置荧光显微镜下观察对照组、DMSO+烧伤血清组、GSI+烧伤血清组细胞黏附的细胞数呈递增趋势，3组的细胞数分别为(152.00±21.07)、(265.33±36.83)、(345.67±30.66)个，3组比较差异有统计学意义(F＝31.09，P<0.05)；DMSO+烧伤血清组和GSI+烧伤血清组黏附的细胞数均明显高于对照组，比较差异均有统计学意义(t＝4.626、9.016，P<0.05)；GSI+烧伤血清组黏附的细胞数明显高于DMSO+烧伤血清组，差异有统计学意义(t＝2.903，P<0.05)。

大鼠烧伤后血清中的ICAM-1含量明显上升，烧伤大鼠血清刺激PMVEC可以导致细胞内ICAM-1含量以及分泌到上清中的ICAM-1含量增加，应用GSI阻断Notch信号通路可以增加ICAM-1的产生及表达，可增加PMVEC对人单核细胞系THP-1细胞的黏附作用。

To investigate the effect of Notch signaling pathway on the intercellular adhesion molecule (ICAM)-1 of pulmonary vascular endothelial cell (PMVEC) induced by serum in burned rats.

Among the 42 SD rats (6-8 weeks), 30 were randomly selected and divided into sham injury group(n=15)and burn group(n=15) according to the random number table method. Rats in burn group were immeresed in hot water at 95 ℃ for 18 s, inflicted with 30% total body surface area full-thickness burn on back, and the rats in the sham injury group were immersed in a 37 ℃ water bath for 18 s to simulate injury. At 6, 12, 24, 48, 72 h after injury, 3 rats were randomly selected from the burn group, and blood was collected from the abdominal aorta. Enzyme-linked immunoadsordent assay(ELISA) were used to detected the content of ICAM-1 in rats’ serum. Rats in the sham injury group were subjected to the same test. Six rats of the remaining 12 rats (6-8 weeks) were taken, and 30% total body surface area full-thickness burn was made according to the aforementioned method, and the serum of the burned rats was prepared at 24 h after the injury; the remaining 6 rats were not treated, and the serum of healthy rats was prepared. Marginal pulmonary tissue was harvested from 3 days old 10 SD rats, and the rat PMVEC were culturned with tissue block method. The morphological characteristics of primary cells cultured for 2 and 6 d were observed under an inverted phase contrast microscope, and the cells in primary cultured for 7 d were identified by flow cytometry. The PMVEC cells of the 4th generation at logarithmic growth stage were taken for experiment. The cells were inoculated on 6-well plates, when the cells grew to 80% confluence, they were divided into 3 groups according to the random number table method: the control group, the dimethyl sulfoxide (DMSO)+ burn serum group and the gamma-secretase inhibitor (GSI)+ burn serum group, with 3 wells in each group. Cells in control group were cultured with 10% healthy rat serum, the latter 2 groups added with 3 μL/mL DMSO and 75 μmol/L GSI respectively, and after 24 h of culture, 10% burn rat serum were added to stimulate the culture for 24 h. The content of ICAM-1 in PMVEC supernatant was determined by ELISA, the content of ICAM-1 in PMVEC of each group was detected by flow cytometry, and the protein expression of ICAM-1 was detected by Western blotting, and the relative protein expression was calculated. The cell adhesion ability of PMVEC was detected. Data were processed with one-way ANOVA and LSD-t test.

(1) The serum contents of ICAM-1 in rats of burn group were (19.77±3.03), (22.09±3.65), (22.44±4.04), (25.40±2.51), (26.37±3.07) pg/mL at 6, 12, 24, 48, and 72 h after injury respectively, which were significantly higher than those in the sham injury group [(10.60±1.51), (11.03±1.95), (10.87±0.89), (9.30±0.89), (10.93±1.22)] pg/mL, the differences were statistically significant between the two groups (t=4.699, 4.466, 3.181, 10.490, 8.097; P<0.05). (2) Primary cells grew slowly after 2 d of culture, cells were seen to migrate from the edge of the tissue mass, and cell fusion was observed at 6 d after culture that them showed a cobblestone appearance. Cells in primary cultured for 7 d were identified by flow cytometry, and the results showed that CD31-positive cells accounted for 98.6%, which was determined to be PMVEC. (3) At 24 h after serum stimulation culture, the contents of ICAM-1 in the supernatant of cells in the control group, the DMSO+ burn serum group, and the GSI+ burn serum group were 1.21±0.25, 2.16±0.12 and 3.07 ±0.30, respectively, and the difference was statistically significant among the three groups(F=46.72, P<0.05). Compared with the control group, the content of ICAM-1 in the supernatant of cells in the DMSO+ burn serum group and GSI+ burn serum group was significantly increased, and the differences were statistically significant (t=5.977, 8.274; P<0.05); the content of ICAM-1 in cell supernatant of GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference between the two groups was statistically significant (t=4.847, P<0.05). (4) At 24 h after serum stimulation culture, the mean fluorescence intensity of ICAM-1 in PMVEC of control group, DMSO+ burn serum group and GSI+ burn serum group were 2 582±143, 3 453±204, 4 559±414, respectively, and the difference was statistically significant among the three groups(F=37.84, P<0.05). Compared with the control group, the content of ICAM-1 in cells of DMSO+ burn serum group and GSI+ burn serum group was significantly increased, and the differences were statistically significant (t=6.056, 7.817; P<0.05); the content of ICAM-1 in the GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference was statistically significant between the two groups (t=4.149, P<0.05). (5) At 24 h after serum stimulation culture, the ratios of ICAM-1/GAPDH in the control group, DMSO+ burn serum group, and GSI+ burn serum group were 0.74±0.08, 1.07±0.08, 1.38±0.28, respectively, and the difference was statistically significant among the three groups(F=30.76, P<0.05). The ratios of ICAM-1/GAPDH in the DMSO+ burn serum group and GSI+ burn serum group were significantly higher than that in the control group, and the differences were statistically significant (t=5.182, 6.990; P<0.05). The ICAM-1/GAPDH ratio in the GSI+ burn serum group was significantly higher than that in the DMSO+ burn serum group, and the difference was statistically significant (t=3.770, P<0.05). (6) After PMVEC was incubated with human mononuclear cell line THP-1 cells for 2 h, the number of human mononuclear cell line THP-1 cells adhered to the cells in the control group, DMSO+ burn serum group and GSI+ burn serum group observed under inverted phase contrast fluorescence microscope showed an increasing trend. The number of cells in the three groups were 152.00±21.07, 265.33±36.83, 345.67±30.66, and the difference was statistically significant among the three groups (F=31.09, P<0.05). The number of adherent cells in the DMSO+ burn serum group and GSI+ burn serum group were significantly higher than that in the control group, and the differences were statistically significant (t=4.626, 9.016; P<0.05); the number of adherent cells in the GSI+ burn serum group was significantly higher than that in DMSO+ burn serum group, the difference was statistically significant (t=2.903, P<0.05).

The content of ICAM-1 in the serum of rats after burn is significantly increased. The stimulation of PMVEC in burn rat serum can lead to an increase in the intracellular ICAM-1 content and the secretion of ICAM-1 into the supernatant. The application of GSI to block Notch signaling pathway can increase the production and expression of ICAM-1 and increase the adhesion of PMVEC to human mononuclear cell line THP-1 cells.