| Objects Firstly to explore impaction on rat liver cell line BRL-3A proliferation exerted by TGF-β1, further use lentivirus expressing shRNA targeted to smad3 gene to silent SMAD3 of BRL-3 A,which is an important downstream protein of TGF-β1/smads pathway, then survey proliferation difference between BRL-3A and BRL-3 A infected by lentivirus again,besides study its action mechanisms preliminary, laying foundations for further studies of accelerating liver regeneration based on TGF-β1 pathway.Methods (1)BRL-3A cells were divided into six groups and exposed to different concentrations of TGF-β1(0,2,4,6,8,10 ng/ml), and cell proliferation was detected by MTT method at 24,36 and 48 hours later, respectively. (2) Set up experimental and control group with or without treatment of TGF-β1(8ng/ml) to conduct the following experiments:①Flow cytometry was performed to measure the cell cycle and apoptosis rate at 24,36 and 48 hours after treatment;②Real-Time Quantitative RT-PCR was used to quantify the expression of cyclinE, cdk-2, EGF, HGF, bcl-2, c-myc, MMP9, NF-κB at mRNA level relatively at the same three time points. (3) Lentivirus vector was packaged and tittered, expressing shRNA targeted to smad3 gene, which then would be used to infect BRL-3A at an MOI of 5.Its founction of silencing SMAD3 in BRL-3A was identified by Real-Time Quantitative RT-PCR and Western-blot at mRNA and protein levels respectively. (4) Cell proliferation of normal BRL-3A and BRL-3A infected by lenvirus was detected by MTT method, meanwhile, cell cycle was measured by flow cytometry, likewise, Real-Time Quantitative RT-PCR was to used to quantify their relative expression of cyclinE, cdk-2, EGF, HGF, bcl-2, c-myc, MMP9, NF-κB at mRNA level.Results (1) MTT proliferation assays revealed that there were no statistical distinctions during those six groups at the three time points; (2)Analysis results from flow cytometry presented that although apoptosis rates of the group with treatment of TGF-β1 were higher than the other, disparities of apoptosis rates or cell cycle at three time points were still not be sured statistically; (3) Real-Time Quantitative RT-PCR found out that compared to control,CyclinEmRNA, Cdk2mRNA, EGFmRNA decreased to 0.194±0.103,0.181±0.064,0.634±0.116 fold at 24 hours, as well as 0.379±0.173,0.457±0.123,0.619±0.112 fold at 36 hours, but increased to 1.956±0.215,2.17±0.471,7.66±0.437 fold at 48 hours; HGFmRNA declined to 0.152±0.068, 0.146±0.053,0.158±0.061 fold respectively at the three time points; Bcl-2 mRNA remained no evident changes at 24,36 hours, while increased to 1.567±0.115 fold at 48 hours; C-myc mRNA,MMP9 mRNA, NF-κB mRNA were upregulated to 1.742±0.389,3.484±0.411, 1.625±0.369 fold at 24 hours,2.292±0.361,1.563±0.323,1.486±0.494 fold at 36 hours, 2.499±0.475,2.233±0.493,3.612±0.364 fold at 48 hours. All the differences of RT-PCR report were statistically significant(P<0.05); (4)We obtained lentivirus vector by packaging and titering,which had infectious activity and high titer of 2.75×10'Tu/ml. Its transfection efficiency was 98.59% at an MOI of 5. Real-Time Quantitative RT-PCR showed that compared to normal BRL-3A, Smad3mRNA of BRL-3A infected by lentivirus decreased to 0.340±0.153 fold (t=17.169,P=0.000).Western-blot revealed that SMAD3 of BRL-3A infected by lentivirus was much lower than normal BRL-3A with statistical significance (t=7.042, P=0.002); (5) MTT and flow cytometry assays inspected that there were no statistical differences in absorbances or cell cycle between these two groups. Real-Time Quantitative RT-PCR found out that compared to normal BRL-3A, CyclinEmRNA, Cdk-2mRNA, MMP9mRNA of BRL-3A infected by lentivirus declined to 0.093±0.032 fold (P=0.030),0.083±0.02 fold (P=0.002),0.039±0.005 fold (P=0.001) respectively, but EGFmRNA, HGFmRNA, Bcl-2mRNA, C-mycmRNA, NF-KBmRNA had no statistical changes.Conclusions Sensitivity of BRL-3A to TGF-β1 is low at the aspects of inducing apoptosis and cell cycle arrest, which may be related to activation of non-smads way, upregulation of NF-κB, Bcl-2, C-myc, MMP9 expression. After silencing SMAD3,which is an important downstream protein of TGF-β1/Smads pathway, BRL-3A proliferated just as normal one, NF-κB, Bcl-2, C-myc expression remained no evident changes, supplying another evidence for that non-Smads way is an important pathway by which TGF-(31 affect BRL-3A proliferation.
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