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目的了解内毒素受体在肝星状细胞活化中的变化和作用。方法分离正常大鼠的肝星状细胞,以逆转录聚合酶链反应法检测其在体外培养过程中内毒素受体(CD14和TLR4)mRNA的表达变化。以细胞免疫染色法检测肝星状细胞内毒素受体CD14的表达。制作肝纤维化和肝硬化的大鼠模型,免疫组织化学法动态检测肝组织内CD14和α平滑肌肌动蛋白的表达变化和定位。结果初分离的肝星状细胞表达低水平的CD14 mRNA,不表达TLR4 mRNA,培养活化的肝星状细胞内毒素受体的表达增强,内毒素可上调这种表达。体外培养10d的肝星状细胞表达CD14蛋白,内毒素作用后CD14表达更明显。在肝纤维化的发展过程中,肝组织内CD14阳性细胞增多,阳性细胞多分布于肝窦周围,晚期CD14阳性细胞聚集在纤维隔内,与α平滑肌肌动蛋白阳性细胞的分布一致。结论肝星状细胞在体内外的活化过程中内毒素受体的表达增强,因此,内毒素受体可能参与肝星状细胞在肝脏炎症和纤维化中的作用。
Objective To understand the changes and roles of endotoxin receptors in the activation of hepatic stellate cells. Methods Hepatic stellate cells of normal rats were isolated and the expressions of endotoxin receptor (CD14 and TLR4) mRNA in cultured rat hepatic stellate cells were detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of endotoxin receptor CD14 in hepatic stellate cells was detected by immunocytochemistry. Rat models of liver fibrosis and cirrhosis were made and the expression and localization of CD14 and α smooth muscle actin in liver tissues were dynamically detected by immunohistochemistry. Results Primary hepatic stellate cells expressed low level of CD14 mRNA but not TLR4 mRNA. The expression of endotoxin receptor in cultured activated stellate cells was enhanced, and endotoxin up-regulated this expression. CD14 protein was expressed in hepatic stellate cells cultured in vitro for 10 days, and CD14 expression was more obvious after endotoxin treatment. During the development of hepatic fibrosis, the number of CD14 positive cells increased, the positive cells distributed around the hepatic sinusoid, and the late CD14 positive cells gathered within the fibrous septum, which was consistent with the distribution of α smooth muscle actin positive cells. Conclusion The expression of endotoxin receptors is enhanced during hepatic stellate cells activation in vitro and in vivo. Therefore, endotoxin receptors may be involved in the hepatic stellate cells in the inflammation and fibrosis.