组织因子在下肢深静脉血栓动物模型中的表达变化及其作用机制

Expression changes and mechanism of tissue factors in animal models of deep venous thrombosis of lower limbs

目的构建大鼠下肢深静脉血栓(DVT)模型,探讨组织因子(TF)表达及作用机制。方法构建TF小干扰RNA(siRNA)特异性沉默TF表达,转染大鼠成纤维细胞RFL-6,反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)实验检测转染效率。通过大鼠尾静脉注射进行动物预处理及分组,72只雄性6~8周龄无特定病原体(SPF)级SD大鼠采用随机数字表法随机分为空白对照组(6只)、假手术组(6只)、模型组(30只)、抑制组(30只),空白对照组不进行任何处理,假手术组仅进行开腹手术,不结扎静脉,模型组行开腹手术结扎静脉构建DVT模型,抑制组在模型组基础上行TF siRNA预处理。分别于术后2、8、24、48、72 h处死大鼠,取血清样本,RT-PCR实验检测不同组别大鼠血样中TF、组织因子途径抑制物(TFPI)、P选择素(P-selection)及P选择素糖蛋白配体1(PSGL-1)mRNA表达水平;酶联免疫吸附试验检测大鼠血清TF、TFPI、P-selection、PSGL-1表达水平,多组数据间比较采用单因素方差分析,两组数据间均数比较采用SNK-q检验;Spearman检验分析TF与各生物学指标在血栓形成中的关系。结果模型组大鼠术后静脉血中TF、TFPI、P-selection、PSGL-1 mRNA表达水平明显高于空白对照组和假手术组大鼠[TF,1.54±0.12、1.02±0.03、1.03±0.02(t=11.322,P<0.05);TFPI,1.47±0.21、1.04±0.03、1.03±0.04(t=9.382,P<0.05);P-selection,1.59±0.12、0.95±0.05、1.03±0.03(t=12.720,P<0.05);PSGL-1,1.47±0.14、0.96±0.03、1.08±0.04(t=10.243,P<0.05)],差异均有统计学意义,模型组大鼠术后静脉血中TF、TFPI、P-selection、PSGL-1表达量均于空白对照组和假手术组大鼠[TF,(102.42±8.42)、(56.32±5.64)、(52.43±6.32)ng/L(t=14.230,P<0.05);TFPI,(42.43±4.45)、(36.43±3.42)、(38.43±4.10)μg/L(t=10.188,P<0.05);P-selection,(34.32±4.43)、(21.32±3.24)、(23.43±3.20)μg/L(t=9.272,P<0.05);PSGL-1,(1432.43±110.42)、(832.43±32.43)、(845.43±43.84)μg/L(t=10.054,P<0.05)],差异均有统计学意义,且模型组大鼠术后2、8、24 h时静血中TF、TFPI、P-selection、PSGL-1 mRNA表达水平明显高于空白对照组和假手术组大鼠[TF,1.54±0.12(t=7.282,P<0.05),1.61±0.21(t=6.640,P<0.05),1.97±0.22(t=8.022,P<0.05);TFPI,1.47±0.21(t=11.022,P<0.05),1.50±0.18(t=12.103,P<0.05),0.52±0.20(t=11.039,P<0.05);P-selection,1.59±0.12(t=11.651,P<0.05),1.68±0.20(t=9.022,P<0.05),2.23±0.31(t=7.292,P<0.05);PSGL-1,1.47±0.14(t=8.023,P<0.05),1.60±0.23(t=7.026,P<0.05),2.20±0.34(t=8.554,P<0.05)],模型组大鼠术后2、8、24 h时静血中TF、TFPI、P-selection、PSGL-1表达量也明显高于空白对照组和假手术组大鼠[TF,(102.42±8.42)ng/L(t=7.166,P<0.05),(135.43±8.53)ng/L(t=9.025,P<0.05),(158.76±22.32)ng/L(t=12.305,P<0.05);TFPI,(42.43±4.45)μg/L(t=15.021,P<0.05),(51.42±5.43)μg/L(t=11.353,P<0.05),(68.64±6.87)μg/L(t=13.004,P<0.05);P-selection,(34.32±4.43)μg/L(t=8.023,P<0.05),(42.43±5.11)μg/L(t=12.020,P<0.05),(59.56±8.31)μg/L(t=10.266,P<0.05);PSGL-1,(1432.43±110.42)μg/L(t=8.102,P<0.05),(1544.32±96.43)μg/L(t=10.442,P<0.05),(1703.42±117.46)μg/L(t=9.225,P<0.05)],差异均有统计学意义,至术后24 h时达最高水平。模型组大鼠术后48 h及72 h静脉血中TF、TFPI、P-selection、PSGL-1 mRNA表达水平出现降低趋势。抑制组大鼠术后2、8、24、48、72 h时静脉血中TF、TFPI、P-selection、PSGL-1 mRNA表达水平明显低于空白对照组及假手术组[TF,0.43±0.01、0.53±0.01、0.59±0.02、0.61±0.03、0.62±0.01、2.02±0.22、2.02±0.24(t=14.385,P<0.05);TFPI,0.45±0.02、0.50±0.02、0.58±0.02、0.60±0.02、0.61±0.03、0.51±0.18、0.50±0.20(t=13.022,P<0.05);P-selection,0.34±0.03、0.55±0.03、0.67±0.04、0.60±0.02、0.55±0.03、2.02±0.16、1.79±0.14(t=15.335,P<0.05);PSGL-1,0.40±0.02、0.54±0.02、0.71±0.04、0.62±0.03、0.57±0.03、1.84±0.16、1.65±0.15(t=10.283,P<0.05)],差异均有统计学意义,抑制组大鼠术后2、8、24、48、72 h时静脉血中TF、TFPI、P-selection、PSGL-1表达量明显低于空白对照组及假手术组[TF,(34.64±5.64)、(50.53±10.54)、(65.43±11.42)、(48.42±12.42)、(38.42±10.24)、(102.32±7.43)、(93.43±6.48)ng/L(t=12.473,P<0.05);TFPI,(21.42±9.40)、(29.42±6.75)、(37.53±5.48)、(32.43±10.48)、(24.54±6.63)、(57.65±6.40)、(53.23±4.30)μg/L(t=11.255,P<0.05);P-selection,(13.42±2.23)、(15.54±3.02)、(18.76±2.43)、(15.43±1.43)、(11.43±2.03)、(43.43±5.03)、(37.65±5.11)μg/L(t=12.336,P<0.05);PSGL-1,(588.54±98.53)、(645.32±103.42)、(785.54±120.32)、(677.76±124.43)、(544.65±109.43)、(1343.53±75.43)、(1197.32±89.32)μg/L(t=8.102,P<0.05)],差异均有统计学意义,抑制组大鼠术后2、8、24 h时各观察指标呈增长趋势,至术后24 h时达最高水平,但静脉血中P-selection、PSGL-1 mRNA表达水平和TF、TFPI、P-selection、PSGL-1表达量均呈现降低表现。相关性分析结果显示,DVT模型中,大鼠静脉血中TF表达水平与TFPI(r=0.632,P<0.05)、P-selection(r=0.513,P<0.05)、PSGL-1(r=0.651,P<0.05)呈明显正相关。结论大鼠DVT模型静脉血中,TF、TFPI、P-selection、PSGL-1表达量与血栓形成进程有关,TF表达与TFPI、P-selection、PSGL-1表达存在正相关,P-selection、PSGL-1可通过介导内皮细胞黏附影响TF表达,调节血栓形成。

Objective To establish a rat model of deep venous thrombosis(DVT),and to explore and analyze the expression changes and mechanism of tissue factor(TF)in animal models.Methods The expression of TF was specifically silenced by TF siRNA.Rat fibroblasts(RFL-6)were transfected,and the transfection efficiency was determined by RT-PCR and Western blotting.Animals through the rat caudal vein injection pretreatment and grouping,6-8 weeks of SPF level 72 male SD rats were randomly divided into blank control group by random number table method(6)and control group(6),model group(30),inhibiting group(30),don′t make any deal with blank control group,control group only laparotomy surgery,not vein ligation model group line laparotomy vein ligation of lower extremity deep vein thrombosis(DVT)model was constructed,inhibit based uplink TF siRNA pretreatment group in the model group.The rats were sacrificed at 2,8,24,48 and 72 hours after operation.Serum samples were taken to detect the mRNA expression levels of TF,tissue factor pathway inhibitor(TFPI),p-selection and p-selectin Glycoprotein Ligand 1(PSGL-1)in blood samples of different groups.The expression levels of TF,TFPI,P-selection and PSGL-1 in serum of rats were detected by ELISA.One-way ANOVA was used to compare the data of multiple groups,and test was used to compare the mean values of data between two groups.Spearman test was used to analyze the relationship between TF and various biological indexes in thrombosis.Results The mRNA expression levels of TF,TFPI,P-selection and PSGL-1 in the venous blood of the model group were significantly higher than those of the blank control group and sham operation group[TF,1.54±0.12,1.02±0.03,1.03±0.02(t=11.322,P<0.05);TFPI,1.47±0.21,1.04±0.03,1.03±0.04(t=9.382,P<0.05);P-selection,1.59±0.12,0.95±0.05,1.03±0.03(t=12.720,P<0.05)].The expression levels of TF,TFPI,P-selection and PSGL-1 in the postoperative venous blood of rats in model group were significantly higher than those in blank control group and sham operation group[TF,(102.42±8.42),(56.32±5.64),(52.43±6.32)ng/L(t=14.230,P<0.05,TFPI,(42.43±4.45),(36.43±3.42),(38.43±4.10)μg/L(t=10.188,P<0.05);P-Selection,(34.32±4.43),(21.32±3.24),(23.43±3.20)μg/L(t=9.272,P<0.05);PSGL-1,(1432.43±110.42),(832.43±32.43),(845.43±43.84)μg/L(t=10.054,P<0.05)],and the mRNA expression levels of TF,TFPI,P-Selection and PSGL-1 in resting blood of rats in model group were significantly higher than those in blank control group and sham operation group at 2,8 and 24 h after operation[TF,1.54±0.12(t=7.282,P<0.05),1.61±0.21(t=6.640,P<0.05),1.97±0.22(t=8.022,P<0.05);TFPI,1.47±0.21(t=11.022,P<0.05),1.50±0.18(t=12.103,P<0.05),0.52±0.20(t=11.039,P<0.05);P-Selection,1.59±0.12(t=11.651,P<0.05),1.68±0.20(t=9.022,P<0.05),2.23±0.31(t=7.292,P<0.05);PSGL-1,1.47±0.14(t=8.023,P<0.05),1.60±0.23(t=7.026,P<0.05),2.20±0.34(t=8.554,P<0.05)],the expression levels of TF,TFPI,P-selection and PSGL-1 in resting blood of rats in model group were also significantly higher than those in blank control group and sham operation group at 2,8 and 24 h after operation[TF,(102.42±8.42)ng/L(t=7.166,P<0.05),(135.43±8.53)ng/L(t=9.025,P<0.05),(158.76±22.32)ng/L(t=12.305,P<0.05);TFPI,(42.43±4.45)μg/L(t=15.021,P<0.05),(51.42±5.43)μg/L(t=11.353,P<0.05),(68.64±6.87)μg/L(t=13.004,P<0.05);P-selection(34.32±4.43)μg/L(t=8.023,P<0.05),(42.43±5.11)μg/L(t=12.020,P<0.05),(59.56±8.31)μg/L(t=10.266,P<0.05);PSGL-1,(1432.43±110.42)μg/L(t=8.102,P<0.05),(1544.32±96.43)μg/L(t=10.442,P<0.05),(1703.42±117.46)μg/L(t=9.225,P<0.05)],and reached the highest level 24 h after operation.The mRNA expression levels of TF,TFPI,P-selection and PSGL-1 in venous blood of model group were decreased 48 h and 72 h after operation.The mRNA expression levels of TF,TFPI,P-selection and PSGL-1 in the venous blood of the inhibited group at 2,8,24,48 and 72 h after operation were significantly lower than those in the blank control group and sham operation group[TF,0.43±0.01,0.53±0.01,0.59±0.02,0.61±0.03,0.62±0.01,2.02±0.22,2.02±0.24(t=14.385,P<0.05));P-selection,0.34±0.03,0.55±0.03,0.67±0.04,0.60±0.02,0.55±0.03,2.02±0.16,1.79±0.14(t=15.335,P<0.05);PSGL-1,0.40±0.02,0.54±0.02,0.71±0.04,0.62±0.03,0.57±0.03,1.84±0.16,1.65±0.15(t=10.283,P<0.05)],the expression levels of TF,TFPI,P-selection and PSGL-1 in venous blood of rats in the inhibited group were significantly lower than those in the blank control group and sham operation group at 2,8,24,48 and 72 h after operation[TF,(34.64±5.64),(50.53±10.54),(65.43±11.42),(48.42±12.42),(102.32±7.43),(93.43±6.48)ng/L(t=12.473,P<0.05).TFPI,(21.42±9.40),(29.42±6.75),(37.53±5.48),(32.43±10.48),(24.54±6.63),(57.65±6.40),(53.23±4.30)μg/L(t=11.255,P<0.05);P-selection,(13.42±2.23),(15.54±3.02),(18.76±2.43),(15.43±1.43),(11.43±2.03),(43.43±5.03),(37.65±5.11)μg/L(t=12.336,P<0.05);PSGL-1,(588.54±98.53),(645,32±103,42),(785.54±120.32),(677.76±124.43),(1343.53±75.43),(1197.32±89.32)μg/L(t=8.102,P<0.05)].The mRNA expression levels of P-selection and PSGL-1 and the expression levels of TF,TFPI,P-selection and PSGL-1 in venous blood were all decreased.Correlation analysis showed that TF expression level in venous blood of the DVT model was positively correlated with TFPI(r=0.632,P<0.05),P-selection(r=0.513,P<0.05)and PSGL-1(r=0.651,P<0.05).Conclusion The expression of TF in venous blood of rat DVT model was positively correlated with the expression of TFPI,P-selection and PSGL-1.P-selection and PSGL-1 can affect the expression of TF and regulate thrombosis by mediating endothelial cell adhesion.