慢病毒介导脯氨酰寡肽酶过表达抑制硫代乙酰胺诱导的大鼠肝纤维化

Lentivirus-mediated prolyl oligopeptidase overexpression attenuates thioacetamide-induced hepatic fibrosis in rats

目的探讨慢病毒介导的脯氨酰寡肽酶(POP)过表达对硫代乙酰胺(TAA)诱导的大鼠肝纤维化模型的影响。方法 40只雄性SD大鼠被随机分为4组,采用5%TAA诱导大鼠肝纤维化模型,各组分别在造模第1 w末门静脉注射生理盐水(正常对照组和TAA模型组)、空病毒(TAA模型+空病毒组)或携POP慢病毒(TAA模型+POP慢病毒组),在造模第4 w末处死动物,留取肝脏组织;采用苏木素-伊红(HE)染色及Masson染色观察肝脏病理形态学变化,采用化学比色法测定肝组织内羟脯氨酸含量,分别采用实时定量PCR或Western blot法检测POP m RNA表达或蛋白水平。结果模型组POP蛋白相对表达量较正常对照组水平显著降低(P<0.05),而POP转基因组POP蛋白水平较其余三组均显著升高(P<0.01);POP m RNA相对表达变化与POP蛋白相一致。TAA模型组和空病毒组的纤维化评分多为Ⅱ级,肝纤维化半定量积分分别为(15.2±1.69)和(15.3±4.62),显著高于正常对照组(1.75土0.63)和POP慢病毒过表达载体组(7.75±2.71)(P<0.05);模型组和空病毒组肝组织羟脯氨酸含量分别为(504.47±111.15)μg/g肝质量和(498.32±90.87)μg/g肝质量,均显著高于正常对照组[(298.20±47.47)μg/g肝质量,P<0.05],而POP转基因组大鼠肝组织中羟脯氨酸含量为(383.52±43.49)μg/g肝质量,显著低于模型组和空病毒组(P<0.05)。结论肝纤维化时肝组织内POP水平下调,慢病毒载体可成功介导POP在大鼠肝组织内过表达,抑制TAA诱导的大鼠肝纤维化,降低肝组织羟脯氨酸水平。

Objective To investigate the effects of lentivirus-mediated prolyl oligopeptidase overexpression on thioacetamide （TAA）-induced liver fibrosis in rats. Methods 40 male Sprague-Dawley （SD） rats were ran-domly divided into four groups. Thioacetamide （TAA,5%） was injected intraperitoneally to induce liver fibrosis in rats. After 1-week TAA/saline treatment,rats were given normal saline （normal control group and TAA model group）,empty virus（TAA plus empty vector group）,or lentivirus containing POP gene（lentivirus-POP）（TAA plus lentivirus-POP group）;Rats were sacrificed 4 weeks after initial TAA/saline treatment;Pathological changes of the liver were examed by HE staining and Masson staining;Hydroxyproline content in liver tissues were detected by chemical colorimetric;Real-time PCR and Western blot were used to determinate POP mRNA expression and POP protein content in liver,respectively. Results Relative level of POP protein in model group was（0.123±0.04）,sig-nificantly decreased compared with normal control [（0.189 ±0.052）,P〈0.05];POP protein level in rats received lentivirus-POP was（0.304±0.044）,significantly higher than that of other groups（P〈0.01）;the changes in POP mR-NA expression was similar with POP protein content. The fibrosis grade in TAA model group and empty virus group were mostly of S2,and the semi-quanti-tative analysis of the fibrotic tissue by Masson staining were （15.2±1.69） and （15.3±4.62）,respec-tively,which were significantly higher than those of the normal controls and TAA plus lentivirus-POP group [（1.75 土 0.63） and（7.75 ±2.71）,P〈0.05];Hy-droxyproline content in model group and empty virus group were[（504.47±111.15）μg/g wet liver weight] and[（498.32±90.87）μg/g wet liver weight],respectively, significantly higher than that of the normal controls [（298.20±47.47）μg/g wet liver weight,P〈0.05],while hydrox-yproline content in TAA plus lentivirus-POP group was [（383.52±43.49）μg/g TAA wet liver weight],significantly lower than those of model group and empty virus group （P〈0.05）. Conclusions POP expression decreases in fi-brotic liver induced by TAA;POP can be successfully overexpressed in rat liver by lentivirus containing POP genes,attenuates TAA-induced liver fibrosis and decreases hydroxyproline content in the rat liver.