Dissertation
Dissertation > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis

The Expression and Significance of IGFBP2、IGFBP6 Induced TGFβ1 in Hepatic Stellate Cell

Author ZhangZuoZuo
Tutor LiuLiXin
School Shanxi Medical
Course Pathology and Pathophysiology
Keywords IGFBP2 IGFBP6 HSC TGFβ1 IGFBP7(rP1) TGFβ1 Collagen I
CLC R575.2
Type Master's thesis
Year 2007
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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.

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