MANF蛋白表达水平与肝纤维化程度的相关性研究

目的：探讨中脑星形胶质细胞源性神经营养因子(MANF)蛋白在慢性乙型肝炎病毒(HBV)感染患者肝纤维化发生发展中的作用及其与临床特点的相关性。方法采用相对和绝对定量同位素标记( ITRAQ)蛋白质组学和免疫组化法检测肝脏穿刺组织中MANF蛋白的表达,分析其表达水平与肝脏炎症纤维化分期的关系；采用实时荧光定量PCR技术检测正常对照( NC)、乙肝病毒携带者( ASC)、慢性乙型肝炎患者( CHB)及乙肝后肝硬化患者( LC)外周血白细胞中MANF mRNA的表达水平,分析其与不同阶段慢性HBV感染者的临床病毒学和生化学指标的相关性。结果 MANF蛋白主要在肝细胞质中表达,其表达水平随着肝脏炎症及纤维化的程度加重而升高。 NC 组、ASC 组和 CHB 组分别与LC组比较,MANF mRNA的表达差异均有统计学意义( P<0．01)。 MANF mRNA 的表达水平在乙肝病毒表面抗原(HBsAg)<1．5×106 IU/L、(1．5×106~2．0×107) IU/L和>2．0×107 IU/L 3组间差异有统计学意义(P<0．05)；在乙肝病毒e抗原( HBeAg)阳性组与阴性组之间差异有统计学意义(P<0．01)；在胆红素正常组与异常组之间差异有统计学意义( P <0．01)。结论 MANF 蛋白可能参与了慢性HBV感染者肝纤维化的发生发展,并与其临床特点相关。

Study on correlation between MANF expression levels and the grade of liver fibrosis

Objective To explore the role of mesencephalic astrocyte-derived neurotrophic factor( MANF) protein in the development of liver fibrosis and clinical significance in the patients with chronic hepatitis B virus ( HBV) infection. Methods The expression of MANF protein in liver biopsy tissues was detected by isobaric tags for rela-tive and absolute quantitation ( ITRAQ) and immunohistochemical method, and then the relationship of MANF ex-pression and the degree of inflammation and fibrosis of liver were analyzed. The levels of MANF mRNA in peripher-al leukocytes were measured by real-time polymerase chain reaction ( PCR) , and analyzed its correlation between clinical virological and biochemical data in asymptomatic hepatitis B virus surface antigen carriers( ASC) , chronic hepatitis B( CHB) , and liver cirrhosis( LC) . Results MANF protein was mainly localized in the cytoplasm of liver cells. The expression levels of MANF were gradually increased with the aggressive inflammation and fibrosis of liv-er. The expression levels of MANF mRNA in the patients with NC, ASC, CHB had a statistically significant differ-ence compared to LC, respectively(P<0. 01). The expression levels of MANF mRNA had a statistically significant difference among three hepatitis B virus surface antigen ( HBsAg) levels groups ( HBsAg<1. 5 × 106 IU/L, 1. 5 × 106 IU/L~2. 0 × 107 IU/L and >2. 0 × 107 IU/L, P<0. 05). The difference of MANF mRNA expression levels was statistical significance between hepatitis B e-antigen ( HBeAg)-positive group and HBeAg-negative group( P<0. 01 ) , between normal and abnormal bilirubin group ( P<0. 01 ) . Conclusion For the patients with HBV infec-tion,MANF protein may be involved in the development of liver fibrosis and assciated with its clinical significance.