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Objective To explore the mechanism of diabetic hepatic fibrosis by observing effects of high glucose and insulin on expressions of transforming growth factor beta 1 (TGF-β1) and tissue inhibitor of metalloproteinase (TIMP-1) mRNAs of hepatic stellate cell (HSC) in rat.Methods Hepatic stellate cell lines in vitro were administrated by different dose of glucose without insulin and glucose with insulin for 72 h,and mannitol was chosen as hyperosmosis control group.Real time fluorescent quantitation polymerase chain reaction (RT-FQ-PCR) was used to determine expressions of TGF-β1 and TIMP-1 mRNAs of HSC in each group.Results Expressions of TGF-β1 and TIMP-1 mRNAs of HSC in each group could be detected and showed no significant difference among groups (P ＞ 0.05).However,in general,expression of TGF-β1 mRNA in glucose with insulin group decreased and TIMP-1 mRNA increased.Conclusions The mRNA expressions of TGF-β1 and TIMP-1 in HSC could not be induced by high glucose alone,high dose of insulin may increase expressions of HSC TIMP-1 and reduce TGF-β1 expression.The main mechanisms of diabetic hepatic fibrosis may not be TGF-β1 pathway but be closely related to TIMP-1 pathway.
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Objective To investigate the effect of microRNA-21 (miR-21) antisense oligonucleoti-de on collagen synthesis in the rat hepatic satellite cells ( HSCs) .Methods Rat hepatic stellate cells were isolated and cultured; the miR-21 antisense oligonucleotide was transfected into HSCs with lipofectamine 2000;after incubation 48 h, the HSCs were collected.The expression of miR-21 was detected with reverse transcription polymerase chain reaction ( RT-PCR) , andα-smooth muscle actin (α-SMA) protein in HSCs with Western blot.The cell proliferation was assayed with methyl thiazolyl tetrazolium ( MTT) method.Re-sults Compared to scrambled control group, the expression of miR-21 was reduced by 76%( P <0.01), the proliferation activity of HSCs was reduced by(26 ±3)%( P <0.01), the expressions of type I and III collagen proteins were reduced by(61 ±7)%and (48 ±6)%( P <0.01).Conclusions The miR-21 an-tisense oligonucleotide could reduce significantly the expression of miR-21, and inhibit HSC proliferation and extracellular matrix production.
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Objective To construct transforming growth factor β receptor Ⅱ (TGFβRⅡ) siRNA expression vector, and investigate the inhibitory effect on TGFβRⅡ in hepatic stellate cells (HSC-T6) ,thus offer a preliminary study for RNAi therapy in liver fibrosis. Methods The two sites of RNAi action were selected in TGFβRⅡ cDNA through online primer design software of Ambion company. The corresponding double-stranded DNA sequence was constructed into pSilencer-U6 plasmid, which could transcribe small interference RNA, the plasmid was transfected into HSC-T6 cells with Lipofectamine 2000. The expression of TGFβRⅡ mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. Results Expression siRNA plasmids of siRNA-a and siRNA-b that target TGFβRⅡ were successfully constructed. Compared with control group, the expression of TGFβRⅡ mRNA was reduced by 0. 89 ± 0. 06 and 0. 25 ± 0. 03 in two siRNA groups, and expression of TGFβRⅡ protein reduced to 0. 86± 0. 05 and 0. 23 ± 0. 02, respectively. TGFβRⅡ expression was inhibited at mRNA and protein levels after transfected with the siRNA-b plasmid( P <0. 01 ).. There was no difference in the TGFβRⅡ expression after transfected with the siRNA-a plasmid( P > 0. 05). Conclusion TGFβRⅡ-siRNA can effective inhibit expression of TGFβRⅡ in HSC-T6.
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Objective To investigate the effect of TGFβ1 siRNA on hepatic satellite cells (HSCs) activation, proliferation and extracellular matrix production. Methods The TGFβ1, siRNA plasmid was transfected into HSCs with Lipofectamine 2000. The supernatant and HSCs were collected after incubation for 72h. The expression of TGFβ1, and a-SMA protein in HSCs was detected by. Western blotting. The expression of type Ⅰ and Ⅲ collagen mRNA was detected by RT-PCR. The cell proliferation was assayed by MTT method. Contents of typeⅣ collagen and hyaluronic acid in supernatant were determined by radioimmuno-assay.Results Compared with scrambled control group, the TGFβ1, and a-SMA protein expression,the activity of HSCs proliferation,the expression of typeⅠ and Ⅲ collagen mRNA,and the contents of type Ⅳ collagen and hyaluronic acid in supernatant were reduced in TGFβ1, siRNA group by (79±5)%,(55±4)%, (25±4)% ,(63±6)% ,(57±4)% ,(53±8)% ,(46±8)% ( P<0.01),respectively.Conclusion TGFβ1, siRNA could significantly reduce the expression of TGFβ1,inhibited HSC activation,proliferation and extracellular matrix production.
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f fibrosis-related factors in LX-2cells were significantly increased after co-cultured with QSG7701-HBx cells, which proved that HBx could induce fibrogenesis in vitro.