Effects Of Antisense TβRI Eukaryotic Expressing Plasmid And That Cooperate With Antisense TIMP-1 Eukaryotic Expressing Plasmid On Rat Liver Fibrosis

Background:Liver fibrosis is a common sequel to diverse liver injuries, and is characteristic of the unbalance of degradation and synthesis of extracellular matrix (ECM). Molecular mechanisms involved in fibrogensis reveal that transforming growth factorβ(TGFβ) and PDGF,especially TGFβ,play a pivotal role in the regulation of the production,degradation and accumulation of extracellular matrix (ECM). At present, the general signal process of TGFβhas already clarified. Signaling by TGFβoccurs through a family of transmember and ser/thr kinase receptors.Both components of the receptor complex,known as receptor I (TβRI) and receptor II (TβRII) are essential for signal transduction. Signaling is initiated by binding of TGFβto the TβRII,once bound,the TGFβ/TβRII complex then recruits the TβRI into a heteromeric complex. Within the heteromeric complex,the kinase domain of the TβRII transphosphorylates and activates the type I receptor kinase,which then functions to propagate the signal to downstream targets, R-Smad. So in theory, the blockage of TGFβsignal transduction through inhibiting the expression of TβRI may have therapeutic effects on liver fibrosis.Matrix metalloproteinases (MMPs) and their inhibitors-tissue inhibitor of matrix metalloproteinases (TIMPs) play an important role in the balance of degradation and synthesis of ECM. In the system the most important pair of balance factors is MMP-1(human)/MMP-13(Rat).In normal liver the two factors keep in equilibrium state. Once the balance is broken, elevating TIMP-1expression can inhibit the degrading activity of MMP-1, and cause the ultra-deposition of ECM.Our present experiments were performed to construct antiseuse TβRI and TIMP-1 eukrayotic expressing plasmids and in vivo transfection.We aimed to test the hypothesis that the introduction of these two exogenous plasmids into a rat model of liver fibrosis may block the action of TGFβand halt the progression of liver fibrosisMETHODS:1.Construction and identification of rat pcDNA3.1(+)-antisense TβRI eukaryotic expressing plasmid TRIzol was performed on the rat liver organization stored in -80℃to obtain the total RNA . The integrality, concentration and purity of total RNA were detected by ultraviolet spectrophotometer and 15g/L agarose electrophoresis. The fragment of TβRI was achieved, and was amplified, by using reversion nested primers-polymerase chain reaction with one step kit and polymerase chain reaction, respectively. Using Cacl2 method induced the susceptibility of cell. The eukaryotic expressing vector, at its multiple cloning sites, as well as the fragment of TβRI, was digested by EcoR I and Xhol I, and then, was purified and retrieved by agarose electrophoresis separation. To construct pcDNA3.1(+)-antisense...