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AIM:To study the effects of Kupffer cell-conditioned medium(KCCN)derived from lipopolysaccharide(LPS)treatment onproliferation of rat hepatic stellate cells(HSC).METHODS:HSC and Kupffer cells were isolated from theliver of Wistar rats by in situ perfusion with pronase andcollagenase and density gradient centrifugation with Nycodenzand cultured.KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)colorimetricassay was used to detect HSC proliferation.The content oftype Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β_1 production in the KCCM was detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM andunstimulated KCCM could significantly promote HSCproliferation[0.132±0.005 and 0.123±0.008 vscontrol group(0.100±0.003),P<0.01],and there was a difference betweenthem(P<0.05).Ten microgram per mililiter LPS-activatedKCCM(0.106±0.010)was unable to promote HSC proliferation(P>0.05).Adding anti-TGF-β_1 antibodies could suppress theproliferation 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).Therewas a parallel behavior between HSC proliferation and increasedECM level.One microgram per mililiter LPS-activated KCCNcontained a larger amount of TGF-β_1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC andKupffer cells described here is simple and reliable.KCCMstimulated by LPS may promote HSC proliferation and collagenaccumulation,which are associated with hepatic fibrogenesis.
AIM: To study the effects of Kupffer cell-conditioned medium (KCCN) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC). METHODS: HSC and Kupffer cells were isolated from theliver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenzand cultured. KCCM was prepared and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β_1 production in the KCCM was detected by enzyme-linked immunosorbent assay (ELISA) .RESULTS: HSC and Kupffer cells isolated had high purity. One microgram per mililiter LPS-activated KCCM and as stimulated KCCM could significantly promote HSC proliferation [0.132 ± 0.005 and 0.123 ± 0.008 vs control group (0.100 ± 0.003, P <0.01), and there was a difference betweenthem (P <0.05) Adding anti-TGF-β 1 antibodies could suppress the proliferation promotion promoted by unstimulated KCCM and LPS (1 μg / ml) -activated KCCM (0.109 ± 0.009) (0.106 ± 0.010) vs 0.123 ± 0.008,0.115 ± 0.008 vs 0.132 ± 0.005, P <0.01). LPS (1 μg / ml or 10 μg / 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 is a parallel behavior between HSC proliferation and increased ECM levels. One microgram per mililiter LPS-activated KCC Ncontained a larger amount of TGF-beta 1 than unstimulated KCCM.CONCLUSION: The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCMstimulated by LPS may promote HSC proliferation and collagenaccumulation, which are associated with hepatic fibrogenesis.