枯否细胞通过组织因子对大鼠急性胰腺炎凝血系统变化的影响

Effects of Kupffer cells on coagulation function of rats with acute pancreatitis through tissue factor

摘要:

目的：观察大鼠急性胰腺炎（acute pancreatitis，AP）组织因子（tissue factor，TF）及凝血系统的变化，探讨枯否细胞（kupf－fer cells，KCs）通过组织因子对大鼠AP凝血系统的作用。方法：108只SD大鼠完全随机分为假手术（sham operation，SO）组、轻症胰腺炎（mild acute pancreatitis，MAP）组、重症胰腺炎（severe acute pancreatitis，SAP）组、SO+氯化钆（GdCl3）组、MAP+GdCl3 组、SAP+GdCl3组。GdCl3预处理组在诱发AP模型前24 h予尾静脉注射GdCl3（10 mg/kg，0.1 ml），分别用1.5%和5%的牛黄胆酸钠胰胆管逆行注射诱导MAP和SAP模型。建模后或假手术组分别于术后 6、12、24 h处死大鼠，检测其血清中淀粉酶及凝血项的变化；ELISA法检测血清中TF的含量；分离并培养 KCs，采用 Western blot 法检测其 TF的蛋白含量；免疫组化检测肝脏中 TF蛋白的表达；HE染色检测胰腺组织病理损伤情况。结果：MAP和 SAP组大鼠血清中淀粉酶含量明显高于 SO组（P＜0.01），与 MAP+GdCl3组、SAP+GdCl3组各相应时间点比较无明显差异。同时间点 MAP组和 SAP组较 SO组血清中TF含量明显升高，MAP+GdCl3组和 SAP+GdCl3组较 MAP组和 SAP组明显降低（P＜0.05）。MAP和 SAP组大鼠肝脏中 TF蛋白表达较 SO组表达明显增加。MAP+GdCl3和 SAP+GdCl3组 KCs中 TF蛋白含量较 MAP组和SAP组明显降低（P＜0.05）。SAP组较 MAP和 SO组大鼠血清凝血酶原时间（prothrombin time，PT）、活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、纤维蛋白原浓度（fibrinogen，FIB）明显升高，SAP+GdCl3组较 SAP明显降低（P＜0.05）。大鼠胰腺病理切片显示，MAP组胰腺间质水肿，SAP组胰腺呈凝固性坏死，伴有大量炎症细胞浸润，MAP+GdCl3和SAP+GdCl3组分别较 MAP和 SAP组病理损伤明显减轻。结论：AP时 KCs 中 TF表达明显增加，并能导致凝血象的改变，抑制 KCs功能可有效降低AP时TF的表达，减少凝血象的改变，从一定程度上可降低AP的发生和发展。

Abstract:

Objective：To observe the changes of tissue factor（TF） and coagulation function in rats with acute pancreatitis（AP） and to explore the role of Kupffer cells（KCs） in cruor function in AP rats via TF. Methods：Totally 108 SD rats are randomly divided into 6 groups including sham operation（SO） group，mild acute pancreatitis（MAP） group，severe acute pancreatitis（SAP） group，SO+gadolinium chloride（Gdcl3） group，MAP+Gdcl3 group，SAP+Gdcl3 group. Rats in Gdcl3 pretreatment group were injected with Gdcl3（10 mg/kg，0.1 ml） through the vena caudalis at 24 h before the establishment of AP model. Rats were retrograde infected with 1.5% or 5% of sodium taurocholate through bile duct to establish MAP model and SAP model. The rats were killed at 6，12，24 h after AP treatment or sham operation. Changes in serum amylase and coagulation makers were detected. The content of TF in serum was measured by ELISA. KCs was isolated and cultured， and then the expression of TF protein was detected by Western blot. The TF expression in liver tissues was measured by immunohistochemistry. The pathological damage of the pancreatic tissue was detected by HE staining. Results：The content of serum amylase was significantly higher in MAP and SAP group than in SO group（P<0.01），while there was no significant difference between MAP group，SAP group and MAP+Gdcl3 group，SAP+Gdcl3 group. The contents of TF markedly increased in MAP and SAP groups compared with those in SO group， while distinctively decreased compared with those in MAP+Gdcl3 and SAP+Gdcl3 group（P<0.05）. The TF protein expressions in the livers of rats in MAP and SAP groups distinctively increased compared with those in SO group. TF protein contents of KCs were obviously lower in MAP+Gdcl3 and SAP+Gdcl3 groups than in MAP and SAP groups（P<0.05）. Plasma concentrations of the prothrombin time（PT），activated partial thrombo-plastin time（APTT） and fibrinogen（FIB） in SAP group obviously increased compared with those in MAP groups and SO groups while markedly decreased in SAP+Gdcl3 group compared with those in SAP group（P<0.05）. The pathological section of rat pancreas showed that the pancreatic interstitial of MAP group was oedematous while the pathological damage of MAP+Gdcl3 group was alleviatived compared with that of MAP group. Pancreas of SAP group was in coagulation necrosis with a large number of inflammatory cells while the pathological damage of SAP+Gdcl3 group was obviously alleviatived compared with that of SAP group. Conclusion：Significant increases of TF expression of KCs leading to the change of coagulation makers after the onset of AP. TF expression and the coagulation can be effectively reduced by inhibiting KCs，which can reduce the occurrence and development of AP to some extent.