| Background/ObjectiveAtherosclerosis(AS) is the leading cause of mortality in the Western world. Increased insight into the mechanisms of atherosclerosis may be close to the relationship between inflammation and coagulation.It is well-established that exposure of tissue factor(TF) to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture,resulting in acute coronary events and myocardial infarction.Some other study of TF possesses a central role in the initiation of inflammation-induced coagulation.The recent realization that the TF pathway is the primary mechanism for activation of coagulation in vivo,and the TF pathway is an attractive therapeutic target,and direct inhibitors against TF or the TF·FVⅡa complex might be of great value.Inneed,iseveral such inhibitors have been tested out in various settings,such as animal models of arterial injury,animal models of DIC,and in in vitro models of thrombus formation.However,in our country there are few studies on it.Therefor, Our study,would construct the recombinant adenovirus vector combined with TF-shRNA and detecte the function on the VSMCs threated by IL-18 in vitro,aims to the value of RNA interference(RNAi) of application in gene therapy and would creat the new motheds of treatment of AS.Methods Contents and ResultsPart one.Construction of expression vector with tissue factor-shRNA plasmid and detection on 293 cellsObjective:1.To confirm the higher transfection reagent and the optimization transfecting siRNA condition.2.To construct the expression vector with TF-shRNA plasmid,transient transfection was used to observe the silence effection of recombinant plasmid of TF gene in 293cells.Methods:1.Using the pEGFP-N1(GFP reportor gene) plasmid combined with the transfection reagents:LipofectamineTM 2000 and lipofectamineTM PLUS,seperately. The small interference RNA directed against human TF was designed and was chemically synthesized with flourecence.Chosed the higher reagent and the higher transfection effect in 293cells.Then,the transfection condition of TF siRNA was optimized in 293cells.2.Small RNA interfering of short hairpin of TF gene (shRNA) was designed,plasmid expression vector with CMV promoter was cloned and recombinant plasmid was constructed.293cells was transfected by lipofectamine TM2000,and the effects of RNA interfering on TF gene expression was observed by RT-PCR.Result:1.LipofectamineTM 2000 was higher transfection to 293 cells than lipofectamineTM-Plus.By optimization,the effects are over 90%,which of transfection TF siRNA to 293s by lipofectamineTM 2000 reagent.2.Successful construction of plasmid expression vector pShuttle-TF-shRNA with siRNA of TF gene hairpin was made,the sequence of heterogeneous gene was proved accurate by using digestion and sequencing.3.RT-PCR method was used to detect mRNA expression of TF gene in 293cells within 24,48,72hours after being transfected by recombinant plasmid,results showed that recombinant plasmid pShuttle-TF-shRNA could remarkably reduce expression level of intracellular TF gene in 24,48,72hours after transfection,compared to that of randomlised control siRNA.Down regulation of mRNA expression level in 293cells was separately 2.11±0.01,1.21±0.05 and 0.56±0.01(F=5976.697,P=0.000) in 24,48 and 72 hours after transfection,and results showed a statistical significance.Conclusion:1.By optimized the transfected condition,the lipofectamineTM 2000 is successful transfection the chemical synthesis TF siRNA with higher effect on 293cells.2.Expression vector with TF-shRNA plasmid is successfully constructed, TFmRNA levels may be remarkably down-regulated by constructed recombinant plasmid,and RNA interfering effects is significant.Part two:Construction,Identification and expression of vector with TF-shRNA recombinant adenovirusObjective:To construct recombinant replication-deficient adenovirus containing TF.Methods:Linearized the shuttle plasmid of CMV promoter(pShuttle-TF-shRNA),in which TF shRNA was inserted,and the adenovirus skeleton plasmid which had conservative gene of adenovirus.Phosphate calcium sediment method was used to tansform two plasmid expression vectors to HEK-293 package cells.Recombinant adenoviruse was achieved by homogenous recombinance of two plasmids in HEK-293 cells.By the construct adenovirous containing TF gene to transfect Hela cells,in order to the biosecurity of recombinant.Result:Defective type of recombinant adenovirus vector was constructed by homogenous recombinance method,augmented and purifed by chloride centrigugation alter sequenceing,final yielde were generally 3×1010pfu/ml.After the transfect the 100 MOI of recombinant adenovirous,the different groups(control group, Ad-GAPDH and Ad-TF)have no showed a statistical significance(P=0.329).Conclusion:Adenovirus vector Ad- TF-shRNA with TF-shRNA is successfully constructed,it is stable,and easy to purify or augment final yielde is high. Part three:In vitro to study the silence of tissue factor gene by recombinant adenovirous-mediated TF-shRNA gene on human vascular smooth muscle cellsObjective:In vitro study was performed to the silence of tissue factor gene by recombinant adenovirous-mediated TF-shRNA gene with the changed by IL-18 on human vascular smooth cell for the gene therapy research on atherosclerosis.Methods:1.The human umbilical arteries vasecular smooth muscle cells(VSMCs)was removed from health umbilical core in bacteria free condition and dissection of VSMCs by attactchment-block culture.Cells were identified by morphological(growth characteristic)and immunological(α-actin) criteria.2.Confirm the best MOI on VSMCs by the directly transfect.The proliferation of VSMCs measured by MTT;VSMCs were infected by recombinant adenovirus in vitro and IL-18 treated.The experimental objects were divided into 3 groups:the control group, the IL-18 treated group,the recombinant Ad-TF group and the Ad-GAPDH group. The expression of TF antigen in cells was measured by ELISA,the TF procoagulant activities by one-time clotting methods,TFmRNA and protein level of different groups were analyzed by RT-PCR and Western Blot methods.Result:1.Characteristic granular cytoplasmic staining pattern were revealed after immunohistochemical labeling forα-actin.All experimental cells are the three and four passaged generations.2.When the 50MOI of recombinant,the transfection of VSMCs is above 80%,when is 100MOI,the transfection is above to 90%.It is suggest that the recombinant adenovirous is high transfection and good for cells.3. The ruslts show there are no significant among the recombinant adenovirous groups when compared to the control group(P＞0.05).4.The TF antigen and procoagulation on VSMCs,which induced by IL-18(100ng/mL),have been increased.When compared to the negative group,there have showed a statistical significance. However,afer treated by recombinant adenovirus Ad-TF-shRNA combined with IL-18,the TF antigen and procoagulant activities have been siginificantly decrease. They show a statistical significance(P=0.000),when comapared to the negative group.TF mRNA gene in VSMCs showed down regulation within 24,48 and 72 hours after transfection by recombinant adenovirus.Protein expression level of intracellular TF gene was remarkably low and with the same of RT-PCR result.Conclusion:1.the recombinant adenovious Ad-TF have no influence on the cells proliferation on VSMCs in vitro.2.The IL-18 have the ability to induced the expression of TF.However,the inducing TF could by siginidicant to reducing by the Ad-TF,which combined the TF shRNA.The stage predominantly mRNA and protein expression of VSMCs is remarkale down after co-treated by IL-18 and recombinant adenovious. |
