| Part I Effect of tissue transglutaminase inhibitor on the expression of FN and Col-Ⅳin human lens epithelial cellsObjective To observe the effect of the tissue transglutaminase(tTG) inhibitor monodansyl cadaverineon(MDC) on the expression of fibronectin(FN) and collagenⅣ(Col-Ⅳ) induced by transforming growth factor-β2 (TGF-β2) in human lens epithelial cells (HLECs).Methods Cultured human lens epithelial cell line(HLE-B3) in vitro were divided into five groups, including:(1)group1, normal control group, (2)group2, 10μg/L TGF-β2-treated group, (3)group3,10μg/L TGF-β2+100μmol/L MDC-treated group, (4)group4,10μg/L TGF-β2+200μmol/L MDC-treated group and (5)group5,10μg/L TGF-β2+400μmol/L MDC-treated group. Semiquantitative RT-PCR was used to assay the expression of tTG, FN and Col-Ⅳin HLE-B3 cells.Results HLE-B3 cells cultured in vitro expressed certain quantity of tTG, FN and Col-Ⅳ. And the expression of them were markedly increased in group2 as compared with that in group 1 (P<0.01). The expression of FN and Col-Ⅳwere significantly inhibited in group3, 4 and 5 by MDC in a concentration-dependent manner as compared with that in group2(P<0.01). Conclusion MDC can inhibit the enhanced expression of FN and Col-IV induced by TGF-β2 in HLECs. tTG may be involved in posterior capsule opacification(PCO) through up-regulating the expression of FN and Col-IV in HLECs.Part II Inhibition of tissue transglutaminase expression in human lens epithelial cells by RNA interferenceObjective To investigate the inhibition of tissue transglutaminase (tTG) expression in human lens epithelial cells(HLECs) by RNA interference.Methods Three specific short interfering RNA (siRNA) of tTG (siRNA-1, siRNA-2, siRNA-3) were designed and synthesized, and transiently transfected into HLE-B3 cells. Real-time RT-PCR and Western blot were used to measure the expression of tTG mRNA and protein respectively.Results after the transfection with siRNA-1, siRNA-2, siRNA-3 for 48 hours, the tTG mRNA expression were decreased to 46.60%,12.84%and 66.75%of that in blank control group respectively, after the transfection with siRNA-1, siRNA-2, siRNA-3 for 60hours, the tTG protein expression were decreased to 60.49%,27.87%and 55.91%of that in blank control group respectively. All differences had statistical significance (P<0.05～P<0.01). The same result was showed in Real-time RT-PCR and Western blot that siRNA-2 had the best inhibition effect on tTG.Conclusion siRNA targeting tTG gene is effective in suppressing the tTG expression. PartⅢThe role of tTG-siRNA in transdifferentiation and extracellular matrix deposition of human lens epithelial cellsObjective To investigate the effect of tissue transglutamiase (tTG) siRNA on transdifferentiation and extracellular matrix deposition of human lens epithelial cells(HLECs) induced by TGF-P2.Methods Cultured HLE-B3 cells in vitro were divided into normal control group, TGF-P2-group and TGF-β2+siRNA-group. Western blot was used to assay the expression of tTG, a-smooth muscle actin(α-SMA), fibronectin(FN), and collagenⅣ(Col-Ⅳ) in HLE-B3 cells.Results The expression of tTG,α-SMA, FN and Col-Ⅳwere markedly increased in TGF-P2-group as compared with that in normal control group (P<0.01). And the expression of tTG,α-SMA, FN and Col-Ⅳwere significantly decreased in TGF-P2+siRNA-group as compared with that in TGF-β2-group (P<0.01).Conclusion siRNA targeting tTG gene can obviously inhibit the synthesis of a-SMA, FN and Col-IV in HLE-B3 cells induced by TGF-β2. It suggests that tTG may play an important role in transdifferentiation and extracellular matrix deposition of HLECs induced by TGF-β2. tTG is hopeful to be a new target for the prevention and treatment of posterior capsule opacification(PCO). |
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