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目的研究与肝纤维化相关miRNA(miR)在日本血吸虫可溶性虫卵抗原刺激小鼠肝细胞(AML12)后的表达情况,为阐明血吸虫感染导致肝纤维化的机制奠定基础。方法使用日本血吸虫可溶性虫卵抗原(Soluble egg antigens,SEA)刺激AML12后,采用定量PCR检测AML12中的miR-122、miR-182、miR-23b、miR-27b及KH型剪切调控蛋白(KHtype splicing regulatory protein,KSRP)的m NA水平,以Western blotting检测KSRP蛋白的表达变化;分别用anti-miR-27b、miR-27b precursor转染AML12后,以定量PCR和Western blotting检测AML12中KSRP的m NA和蛋白水平。结果经SEA刺激后,AML12中miR-182、miR-23b及miR-27b m RNA水平下降(P均<0.05),而细胞中miR-122 m RNA与KSRP的m RNA水平及蛋白表达水平均上调(P均<0.05)。此外,anti-miR-27b与miR-27b precursor转染组KSRP的m RNA水平均无明显变化,但anti-miR-27b组细胞中KSRP的蛋白表达增加,miR-27b precursor组KSRP的蛋白表达降低。结论SEA刺激AML12导致诸多与肝纤维化相关的miRNA及KSRP的表达发生变化,其中miR-27b可调控KSRP的表达,这为进一步研究血吸虫感染导致的肝纤维化的分子机制奠定了基础。
Objective To investigate the expression of miRNA (miR) associated with liver fibrosis induced by Schistosoma japonicum soluble egg antigen in mouse hepatocytes (AML12), and lay a foundation for clarifying the mechanism of hepatic fibrosis caused by schistosoma infection. Methods After AML12 was stimulated with soluble egg antigens (SEA) of Schistosoma japonicum, the levels of miR-122, miR-182, miR-23b, miR-27b and KH type-I The expression of KSRP protein was detected by Western blotting after transfected with AML12 by anti-miR-27b and miR-27b precursor respectively. The mRNA and protein expression of KSRP in AML12 were detected by quantitative PCR and Western blotting NA and protein levels. Results After stimulation with SEA, the levels of miR-182, miR-23b and miR-27b m RNA in AML12 were decreased (all P <0.05), while the mRNA and protein levels of miR-122 m RNA and KSRP were up-regulated (P <0.05). In addition, there was no significant difference in the m RNA levels of KSRP between anti-miR-27b and miR-27b precursor transfection group, however, the protein expression of KSRP in anti-miR-27b group was increased and the protein expression of KSRP in miR-27b precursor group was decreased . Conclusions Stimulation of AML12 by SEA results in many changes of miRNA and KSRP related to hepatic fibrosis. MiR-27b regulates the expression of KSRP, which lays the foundation for further study on the molecular mechanism of hepatic fibrosis induced by schistosome infection.