The Study Of Expression Of IGFBP2, IGFBP4 In Hepatocyte With Experimental Injury

Objective:To explore mechanism of IGFBP2,IGFBP4 in hepatocyte with injury induced TNFa and TGF-β₁.Methods:1.Human hepatocyte line HL-7702 was cultured in vitro and treated with TNF-α(10ng/ml,20ng/ml,30ng/ml,40ng/ml for 24h)or TGFβ₁(2ng/ml,4ng/ml,8ng/ml,16ng/ml for 24 h)and established the normal control group(incubated with equal PBS),then the expression of IGFBP2,4 was detected by immunocyte-chemistry staining.2.HL-7702 was cultured in vitro and treated with TNF-α(10ng/ml,20ng/ml,30ng/ml,40ng/ml for 24h)or TGFβ₁(2ng/ml,4ng/ml,8ng/ml,16ng/ml for 24 h),then inhibition ratio of hepatocyte was detected by MTT assay.3 HL-7702 was cultured in vitro and established the normal control group(incubated with equal PBS)and treated with TNFα20ng/ml group for 48h,then the apoptosis of hepatocyte was detected by both the high specificity Annexin-V /PI and high sensitivity TUNEL assay.Results:1.The results of immunoeyte-ehemisty staining:The groups treated TNF-αor TGFβ₁ had positive staining both in cytoplasm and on cytomembrane showing some buffy particles.The positive staining oflGFBP2,4 in groups treated TNF-αor TGFβ₁ was significantly higher than that in the normal control group.The positive staining of IGFBP2,4 in groups treated 20ng/ml TNF-αor 4ng/ml TGFβ₁ was the strongest among the all groups(Comparison 20ng/ml TNFαwith the control group the expression of IGFBP2 is 40.66±5.80 vs 0.53±0.14;the expression of IGFBP4 is 41.16±9.18 vs 0.32±0.08,P＜0.05.Comparison 4ng/ml TGFβ₁ with the control group the expression of IGFBP2 is 85.48±6.09 vs 0.53±0.14;the expression of IGFBP4 is 82.48±4.00 vs 0.32±0.08,P＜0.05).2.MTT assay The experimental groups treated with TNF-αstatistically significant difference with the control group(inhibitory ratio 10ng/ml: 28.80,20ng/ml:34.80,30ng/ml:19.60,40ng/ml:18.40,p＜0.05).The experimental groups treated with TGFβ₁ statistically significant difference with the control group(inhibitory ratio 2ng/ml:2.60,4ng/ml:18.90,8ng/ml:8.24,16ng/ml:10.86,p＜0.05).3.Correlationanalysis There was a positive correlation between IGFBP2,4 and inhibitory ratio of hepatocyte treated TNF-αor TGFβ₁(IGFBP2 induced TNFαand inhibitory ratio:r= 0.497(P＜0.05);IGFBP4 induced TNFαand inhibitory ratio:r=0.534(P＜0.05).IGFBP2 induced TGFβ₁ and inhibitory ratio:r=0.642(P＜0.01);IGFBP4 induced TGFβ₁ and inhibitory ratio: r=0.600(P＜0.01)).4.Apoptosis detected flow eytometry with Annexin/PI Compared with normal control group,the experimental groups markedly increased in the percentage of apoptosis. TNF-α(7.86±1.10 vs.4.76±1.11,P＜0.01).5.TUNEL assay Nuclei stained buffy was detected as positive cells which show typical morphological changes of apoptotic cells.That is the cells changed into smaller,rounded and nuclear condensation.Compared with normal control group, the experimental groups markedly increased in the rate of apoptosis(21.13±4.61vs3.25±1.50,P＜0.01).Conclusion:Expression of IGFBP2,4 was markedly increased while human hepatocyte was injuried by TNF-αor TGFβ₁ and there was a positive correlation between IGFBP2,4 and inhibitory rate of hepatocyte.This study reaffirms the presumption that IGFBP2,4 may be involved in the process of hepatocyte with injury,it may play an important role in hepatocyte with injury mediated TNF-αor TGFβ₁.