Establishment, Functional Study And Intervention By Compound Astragalus And Salvia Miltiorrhiza Extract Of Three Plasmids Including Smad3 WT, Smad3 EPSM And Smad3 3S-A Stably Transfected HepG2 Cell Lines

ObjectivesTo construct three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines by liposome transfection method.1. To observe the effects of transfection three plasmids on morphology and proliferation of HepG2 cells2. To investigate the effects of alternative up-regulation of pSmad3C or pSmad3L protein expression on proliferation, cell cycle and apoptosis in HepG2 cells.3. To investigate the effects of high concentration CASE (80 mg/L) on alternative up-regulation of pSmad3C or pSmad3L protein expression on proliferation and apoptosis in HepG2 cells.Methods1. cell culture and treatmentHepG2 cells were cultured in serum-free medium for 24 h with or without CASE (20, 40,80mg/L) treatment, subsequently the cells were treated with TGF-β1 for the determined concentrations and time depending on the different purposes. The cells of the control group were added to an equal volume of vehicle.2. Establishment stably transfected with three plasmids Smad3 WT, Smad3 EPSM, Smad3 3S-A of HepG2 Cell LinesThree plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A were respectively transfected into HepG2 cells by Liposome transfection reagent, positive cells screened by G418 (600μg/ml) via co-culture for two weeks, than maintained by G418 (300 μg/ml) for four weeks, obtain stable cell lines and expand the training.3. The aim protein expression of stable cell lines by western-blot and immunofluorescence assay.3.1 Western-blotHepG2 and stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A cell lines external culture, after incubation,cells were harvested and lysed for total protein extraction. The expression of Smad3, pSmad3C, pSmad3L protein was measured by western-blot.3.2 ImmunofluorescenceHepG2 and stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A cell lines were seeded on sterile slides in 24-well plates. After incubation, the pretreated cells were fixed and blocked. The incubated with each primary antibody overnight followed by incubation with fluorescein isothiocyanate-conjugated IgG Afterwords, the slides were viewed and photographed under a fluorescence microscope to observe the intracellular localization of phosphorylated Smad3.4. Effect of stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A and intervened by CASE on HepG2 cell proliferationHepG2 and stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A cell lines external culture, After starving in the serum-free medium overnight, the CASE groups were giving the corresponding concentrations of drugs for 24 h, the TGF-β1 gropes added 9 pmol TGF-β1 stimulate for 16 h and the control group cells adds the equal volume serum-free medium, optical density was detected at 570 nm by microplate reader.5. Effect of stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A and intervened by CASE on HepG2 cell cycle and apoptosisHepG2 and stably transfected three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A cell lines external culture, After starving in the serum-free medium overnight, the CASE groups were giving the corresponding concentrations of drugs for 24 h, the TGF-β1 gropes added 9 pmol TGF-β1 stimulate for 1 h and the control group cells adds the equal volume serum-free medium, the cell cycle and apooptpsis was detected by FCM (flow cytometry)Results1. Western-blot and immunofluorescence rusults showed that comparing with the control Smad3 protein expression was up-regulated in separately transfected Smad3 WT, Smad3 EPSM and Smad3 3S-A HepG2 cell lines. The corresponding pSmad3C or pSmad3L protein expression were up-regulated in separately transfected Smad3 WT, Smad3 EPSM and Smad3 3S-A HepG2 cell lines induced by TGF-β1.2. The results from MTT experiment showed that TGF-β1 could induced proliferation of HepG2 cells with or without the transfection of Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids, meanwhile transfected Smad3 EPSM plasmids could significantly inhibit proliferation of HepG2 cells induced by TGF-β1, transfected Smad3 3S-A plasmids accelerate proliferation of HepG2 cells induced by TGF-β1 (P<0.05). The cell proliferation was inhibited in a dose-dependent manner by CASE (20、40、80mg/L) treatment induced by TGF-β1(P<0.01), and the transfected Smad3 EPSM plasmids HepG2 cells proliferation inhibition was obviously stronger comparing with the control, transfected Smad3 WT and Smad3 3S-A plasmids HepG2 cells (P<0.05).3. Cell cycle analysis showed that the G0/G1 phase of HepG2 cells with stable transfection of Smad3 EPSM plasmid increased compared with HepG2 cells with or without stable transfection of Smad3 WT plasmid, meanwhile the G2/M phase of HepG2 cells with stable transfection of Smad3 3S-A plasmid increased induced by TGF-β1(P<0.01). The cell apoptosis analysis showed that compared with HepG2 cells with or without stable transfection of Smad3 WT plasmid, apoptosis rate of Smad3 EPSM stably transfected HepG2 cell line was dramatically increased, while apoptosis rate of Smad3 EPSM stably transfected HepG2 cell line observably decreased (P< 0.01). The cell apoptosis was promoted by high concentration CASE (80 mg/L)treatment (P<0.01), and the transfected Smad3 EPSM plasmids HepG2 cells apoptosis promoted was obviously higher comparing with the transfected Smad3 WT and Smad3 3S-A plasmids HepG2 cells. (P<0.05, P<0.01)Conclusions1. The three plasmids of Smad3 WT, Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines have been successively constructed.2. To explore the confirmed alternative up-regulation of pSmad3C protein expression inhibits cell proliferation, induce cell block in G0/G1 phase and promote cell apoptosis, while alternative up-regulation of pSmad3L protein expression promote cell proliferation, drive cell cycle and inhibits cell apoptosis in HepG2 cells.3. The cell proliferation was inhibited in a dose-dependent manner by CASE (20、40、 80mg/L) treatment induced by TGF-β1, and proliferation inhibition the pSmad3C protein high expressionwas obviously higher than the pSmad3L protein high expression in HepG2 cells.4.The cell apoptosis was promoted by high concentration CASE (80 mg/L) treatment, and apoptosis effected the pSmad3C protein high expressionwas obviously higher than the pSmad3L protein high expression in HepG2 cells.