肝细胞提取物生物学活性研究

为深入研究肝细胞提取物的生物学活性,以正常人肝细胞株、肝癌细胞株、肝星状细胞株及原代培养的肝星状细胞为研究靶细胞,观察在不同浓度肝细胞提取物作用下,上述细胞的增殖活性(MTT法)、细胞凋亡活性、细胞外基质成分透明质酸(HA)产生水平及I型胶原基因启动子活性的影响,同时利用流式细胞术比较正常成人外周血单个核细胞(PBMC)在肝细胞提取物作用下,活性T淋巴细胞(CD4+/CD69+)的变化。结果发现肝细胞提取物对正常人肝细胞系(张氏肝细胞)具有明确刺激增殖作用,而对原代培养的肝星状细胞具有抑制增殖作用;肝星状细胞株HSC-T6的研究中,肝细胞提取物具有抑制其细胞外基质成分HA产生、抑制α1(I)型胶原基因启动转录的活性。60μg/ml的肝细胞提取物对肝癌细胞株Hep G2具有明确的诱导凋亡作用。正常成人PBMC经肝细胞提取物作用后活性T淋巴细胞(CD4+/CD69+)数量显著上升。研究表明,肝细胞提取物具有促进肝细胞再生、抑制肝纤维化发生和抗肿瘤作用,并首次发现具有免疫调节作用。

Study on biological effects of liver cell extract

To study the biological effect of liver cell extract in vitro,normal human hepatocytes cell line(Zhang's hepatocyte),passaged primary rat hepatic stellate cells(HSC) called myofibroblasts(MFB),rat hepatic stellate cell line(HSC-T6) and hepatoma cell line(HepG2) were studied with respect to proliferation,extracellular matrix production and apoptotic activities after treatment with various amount of extract for 24~48 hours by MTT,RIA,gene transfection and reporter gene analysis.Flow cytometry was used for quantitation of CD4~(+)/CD69~(+) lymphocytes in normal human peripheral blood mononuclear cells(PBMC) with or without liver extract treatment.It was demonstrated that strong stimulatory proliferation effect was evident in Zhang's hepatocytes,while inhibitory effect was found in MFB from primary rat HSC.Hyaluronic acid(HA) concentration in HSC-T6 culture supernatant as well as reporter gene activities driven by α1(I) procollagen gene promoters in HSC-T6 was significantly decreased after treatment with the extract.Fluo-Anexin V binding apoptotic HepG2 cells were more prominent if 60 μg/ml extract presented.More active T lymphocytes with CD4~(+)/CD69~(+) positive markers existed in the presence of the extract.In the present study,liver cell extract is effective for hepatocyte regeneration,antifibrogenesis as well as hepatoma cell inhibition in vitro.The improvement of T lymphocytes function by cell extract is reported here for the first time,which is helpful for immune surveillance.