The Expression And Significance Of IGFBP2,IGFBP6 Induced TGFβ₁ In Hepatic Stellate Cell

Objective:To evaluate the change in the expression of IGFBP2,IGFBP6 induced TGFβ₁ in hepatic stellate cell,and to investigate the effect of IGFBP2,6 on human with liver fibrosis.Method:Rat HSC-T6 were cultured in vitro and established the normal control group (incubated with equal PBS)and TGFβ₁ treatment with 2ng/ml group,4ng/ml group,8ng/ml group,16ng/ml group for 24h,cell-coated dishes were attained or cytoplasmic proteins were extracted,then the expression of IGFBP2,6 were detected by both immunocyte-chemistry staining and Western Blot.Result:1)The results of immunocyte-chemisty staining:Each group had positive staining in cytoplasm showing some buffy particles.The positive staining of IGFBP2,IGFBP6 in TGFβ₁ treatment with 2ng/ml group(4.22±0.36,1.99±0.16),4ng/ml group(5.42±0.36,5.18±0.32),8ng/mlgroup(4.94±0.33,4.02±0.33),16ng/mlgroup(3.99±0.31,3.07±0.32) were significantly higher than that in the normal control group(2.26±0.29,5.17±0.41)(P＜0.05),and there was a dose-dependent relationship in a certain range and the positive staining of IGFBP2,IGFBP6 in TGFβ₁ treatment with 4ng/ml group was the most strong among the total groups(P＜0.05).2)The results of Western Blot analysis:The place of 34kD,56kD,43kD represented IGFBP2,IGFBP6 andβ-actin respectively.TGFβ₁ treatment with 2ng/ml group,4ng/ml group,8ng/ml group,16ng/ml group all had obvious expression of protein straps,normal control group in IGFBP2 saw no straps,only in IGFBP6 had a minute strap.After corrected byβ-actin,IGFBP2 protein in TGFβ₁ treatment with 2ng/ml group (0.9901±0.0054),4ng/ml group(1.0335±0.0078),8ng/ml group(0.9847±0.0157),16ng/ml group(0.7465±0.0132)were significantly higher than that in the normal control group(0.0331±0.0079)(P＜0.05),IGFBP6 protein in TGFβ₁ treatment with 2ng/ml group (1.0244±0.0171),4ng/ml group(1.0379±0.0151)were significantly higher than that in the normal control group(1.0085±0.0131)(P＜0.05),and TGFβ₁ treatment with 4ng/ml group the expression of IGFBP2,IGFBP6 was the most strong among the total groups(P＜0.05). Conclusion:The expression of IGFBP2,IGFBP6 induced TGFβ₁ that has been recognized as the strongest leading liver fibrosis factor in HSC-T6 significantly increased,indicating IGFBP2,IGFBP6 may involved in the formation of liver fibrosis. Objective:To study the expression of IGFBP7(rP1)induced TGFβ₁ and the influence of anti-IGFBP7(rP1)antibody on the production of Collagen I induced TGFβ₁ in HSC,and to investigate the effect of IGFBP 7(rP1)on human with liver fibrosis.Method:Rat HSC-T6 were cultured in vitro and established the normal control group (incubated with equal PBS)and TGFβ₁ treatment with 2ng/ml group,4ng/ml group,8ng/ml group,16ng/ml group for 24h,cell-coated dishes were attained or cytoplasmic proteins were extracted,then the expression of IGFBP7(rP1)were detected by both immunocyte-chemistry staining and Western Blot.Simultaneously,to collect the supematant in HSC-T6 treatment with TGFβ₁ 2ng/ml,4ng/ml,anti-IGFBP7(rP1)antibody 0.1ug/ml,anti-IGFBP7(rP1)antibody lug/ml,anti-IGFBP7(rP1)antibody 2ug/ml,TGFβ₁ 2ng/ml+anti-IGFBP(rP1)7 antibody 0.1ug/ml,TGFβ₁ 2ng/ml+anti-IGFBP7(rP1)antibody lug/ml,TGFβ₁ 2ng/ml+ anti-IGFBP7(rP1)antibody 2ug/ml,TGFβ₁ 4ng/ml+anti-IGFBP7(rP1)antibody 0.1ug/ml,TGFβ₁ 4ng/ml+anti-IGFBP7(rP1)antibody lug/ml for 24h,and measure the content of collagen I by ELISA.Result:1)The results of immunocyte-chemisty staining:groups treated by TGFβ₁ had positive staining in cytoplasm showing some buffy particles.The positive staining of IGFBP7(rP1)in TGFβ₁ treatment with 2ng/ml group(3.71±0.32),4ng/ml group(6.45±0.33),8ng/ml group(4.91±0.31),16ng/ml group(4.31±0.26)were significantly higher than that in the normal control group(3.71±0.32)(P＜0.05),and there was a dose-dependent relationship in a certain range and the positive staining of IGFBP7(rP1)in TGFβ₁ treatment with 4ng/ml group was the most strong among the total groups(P＜0.05).2)The results of Western Blot analysis:The place of 31kD,43kD represented IGFBP7(rP1)andβ-actin respectively.TGFβ₁ treatment with 2ng/ml group,4ng/ml group,8ng/ml group,16ng/ml group all had obvious expression of protein straps,normal control group in IGFBP7(rP1)saw no straps.After corrected byβ-actin,IGFBP7(rP1)protein in TGFβ₁ treatment with 2ng/ml group (1.0464±0.0317),4ng/ml group(1.1575±0.0220),8ng/ml group(1.0060±0.0225),16ng/ml group(1.0585±0.0215)were significantly higher than that in the normal control group(0.0288±0.0113)(P＜0.05),and TGFβ₁ treatment with 4ng/ml group the expression of IGFBP7(rP1)was the most strong among the total groups(P＜0.05).This result was in accord with the result of immunocyte-chemistry staining.3)The results of ELISA:The content of collagen I in TGFβ₁ treatment with 2ng/ml group(41.93±2.95),4ng/ml group(49.44±3.21) were significantly higher than that in the normal control group(36.61±1.28)(P＜0.05).The content of collagen I in TGFβ₁ treatment with 2ng/ml group(41.93±2.95)was significantly lower than TGFβ₁ treatment with 4ng/ml group(49.44±3.21)(P＜0.05).There were no significant differences between the normal control group(36.61±1.28)and anti-IGFBP7(rP1)antibody0.1 ug/ml group(35.46±5.80),anti-IGFBP7(rP1)antibody 1 ug/ml group(36.38±2.59),anti-IGFBP7(rP1)antibody 2ug/ml group(37.26±2.12)(P＞0.05).The content of collagen I in TGFβ₁ 2ng/ml+anti-IGFBP7(rP1)antibody 0.1ug/ml group(38.24±2.76),TGFβ₁ 2ng/ml+anti-IGFBP7(rP1)antibody lug/ml group(33.95±3.27),TGFβ₁ 2ng/ml+anti-IGFBP7(rP1)antibody 2ug/ml group(34.59±3.97)were significantly lower than TGFβ₁ 2ng/ml group(41.93±2.95)(P＜0.05),but no differences than the normal control group(36.61±1.28)(P＞0.05).There were no significant differences among TGFβ₁ 2ng/ml+anti-IGFBP7(rP1)antibody 0.1ug/ml group(38.24±2.76),TGFβ₁2ng/ml+ anti-IGFBP7(rP1)antibody 1ug/ml group(33.95±3.27),TGFβ₁ 2ng/ml+anti-IGFBP7(rP1) antibody 2ug/ml group(34.59±3.97)(P＞0.05).The content of collagen I in TGFβ₁ 4ng/ml+anti-IGFBP7(rP1)antibody 0.1ug/ml group(40.24±3.46),TGFβ₁ 4ng/ml+ anti-IGFBP7(rP1)antibody 1ug/ml group(32.61±4.86)were significantly lower than TGFβ₁ 4ng/ml group(49.44±3.21)(P＜0.05),but no differences than the normal control group (36.61±1.28)(P＞0.05).The content of collagen I in TGFβ₁ 4ng/ml+anti-IGFBP7(rP1)antibody 1ug/ml group(32.61±4.86)was significantly higher than TGFβ₁ 4ng/ml +anti-IGFBP7(rP1)antibody 0.1ug/ml group(40.24±3.46)(P＜0.05).4)The results of correlation analysis:There was a positive correlation between IGFBP7(rP1)and collagen I in HSC-T6 treated TGFβ₁(r=0.833,P＜0.01).Conclusion:1,The expression of IGFBP7(rP1)induced by TGFβ₁ that has been considered as the most strong factor to lead liver fibrosis in HSC-T6 significantly increased.2,There is a positive correlation between IGFBP7(rP1)and collagen I in HSC-T6 treated by TGFβ₁.3,Anti-IGFBP7(rP1)antibody can reverse over-production of collagen I induced by TGFβ₁ in HSC-T6.4,IGFBP7(rP1)involved the information of liver fibrosis and IGFBP7(rP1)played an important role in the emergence and development of liver fibrosis.