AIM:To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).METHODS:HSC and Kupffer cells were isolated from ...AIM:To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).METHODS:HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β1 production in the KCCM was detected by enzymelinked immunosorbent assay (ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132±0.005 and 0.123±0.008 vscontrol group (0.100±0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106±0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-β1 antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS(1μg/ml)-activated KCCM (0.109±0.009 vs 0.123±0.008,0.115±0.008 vs 0.132±0.005, P<0.01). LPS (1μg/ml or 10μg/ml) could not promote HSC proliferation immediately (0.096±0.003 and 0.101±0.004 vs 0.100±0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-β1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.展开更多
文摘AIM:To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).METHODS:HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β1 production in the KCCM was detected by enzymelinked immunosorbent assay (ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132±0.005 and 0.123±0.008 vscontrol group (0.100±0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106±0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-β1 antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS(1μg/ml)-activated KCCM (0.109±0.009 vs 0.123±0.008,0.115±0.008 vs 0.132±0.005, P<0.01). LPS (1μg/ml or 10μg/ml) could not promote HSC proliferation immediately (0.096±0.003 and 0.101±0.004 vs 0.100±0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-β1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.
