The Target Effect Of Retroviral Vector With Tissue-Type Plasminogen Activator Gene On Thrombolysis

Partâ… Construction of recombinant enhanced green fluorescent protein gene retroviral vector with tissue-type plasminogen activator gene and pure packaging cell line producing high titer retrovirusesObjective To construct recombinant enhanced green fluorescent protein (EGFP) gene retroviral vector with human tissue-type plasminogen activator (tPA) gene and culture pure packaging cell line producing high titer retroviruses.Methods tPA gene was amplificated by polymerase chain reaction (PCR) and was cloned into retroviral pLEGFP-N1 plasmid. First, the pLEGFP-N1-tPA was identified by restriction endonuclease digestion,PCR analysis and DNA sequencing. Second, the pLEGFP-N1-tPA was transferred into packaging cell PT67 by gene transfection reagent SofastTM. The PT67/ pLEGFP-N1-tPA cells were selected by the corresponding antibiotics. According to the EGFP, the cell expressing strong EGFP was signed. When the signed cell grew as a bundle cells, they were picked out of the plate and cultured to be a pure packaging cell line/ pLEGFP-N1-tPA cells all expressing EGFP. Last, the virus titer produced by pure packaging cells was assayed using NIH3T3.Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed the recombinant pLEGFP-N1-tPA vector containing tPA gene. The virus titer of the pure packaging cell line PT67/pLEGFP-N1-tPA cells was 1×10⁷CFU(cloning forming unit)/ml.Conclusion We have successfully constructed a kind of recombinant retroviral vector pLEGFP-N1-tPA as well as a pure packaging cell line producing high virus titer. This provide a good basis for the study and application of idea synthetic valves with tPA gene local target therapy.Partâ…¡ECUV304 and heart myocytes were infected by pLEGFP-N1-tPA and the purified ECUV304/pLEGFP-N1-tPA cell line was constructed.Objective To observe thrombolysis of supernatant from purified ECUV304/ pLEGFP-N1-tPA cell line and infected cardio myocytes in vitro thrombi model and tPA expressing.Methods ECUV304 was infected by pLEGFP-N1-tPA and one ECUV304/ pLEGFP-N1-tPA cell expressing strong EGFP was cultured as a bundle. Bundle of ECUV304/pLEGFP-N1-tPA→moved out plate by micro operation to culture as a cell line. Supernatant from purified ECUV304/pLEGFP-N1-tPA cell line with contrast were dropped into vitro thrombi model and thrombolysis were observed. According to the activity of lumbrukinase, tPA activity was accounted. tPA content was measured by ELISA. Western blot checked out exogenous tPA band. Origin cardio myocytes from 1-4d neonate rats were infected by pLEGFP-N1-tPA. tPA was checked as above.Results Supernatant from purified ECUV304/pLEGFP-N1-tPA cell line and infected cardio myocytes had obviously big thrombolysis zone. tPA activity from purified ECUV304/pLEGFP-N1-tPA cell line was 502.16u/10⁶ cells/24h and content of tPA was 716.27ng/10⁶ cells/24 h. Exogenous tPA band was checked out by Western blot. tPA activity from infected cardio myocytes was 464.27u/10⁶ cells/24h and content of tPA was 607.34ng/10⁶ cells/24h. As the same condition, exogenous tPA band was checked out by Western blot.Conclusion We successfully cultured purified ECUV304/pLEGFP-N1-tPA cell line and supernatant from that cell line had obviously thrombolysis. Activity and content of tPA were all high and exogenous tPA band was checked out. Also, supernatant from infected heart muscle cells by pLEGFP-N1-tPA had apparently thrombolysis zone. Activity and content of were high and exogenous tPA band were demonstrated.Partâ…¢Observing thrombolysis on exogenous Dacron patches transplanted in inferior caval vein that transferred pLEGFP- N1-tPA in localObjective To identify the target thrombolysis of recombinant tissue-type plasminogen activator (tPA) transferred into the tissue around the Dacron patch (the same materials making of the ring of mechanical valve) in inferior caval vein in vivo.Methods 70 Dacron patches were transplanted into the inferior caval veins of 70 New Zealand white rabbits. The rabbits were randomly divided into 3 groups according to the different handling methods, including local pLEGFP-N1-tPA transferred group (gene therapy group, 30 animals), pLEGFP-N1 transferred group (control group, 20 animals), DMEM+10% Serum injected group (blank control group, 20 animals). Samples of blood,Dacron pieces and inferior caval veins from half of above in each group were harvested on second day and another half on 75th day after surgery. So we classified the 3 groups into 6 sub-units A₁,B₁,C₁,A₂,B₂,C₂. The EGFP expression of harvested inferior caval veins were observed under the confocal. The thrombi on the surface of Dacron patches were detected by stereoscope and electron microscope. The tPA expression in inferior caval veins were detected by Western blot and their thrombolysis and activities were observed and calculated in plasma plates. ElISA were used to identify the contents of tPA.Results At the second, 75th day, much and strong EGFPs in inferior caval veins of gene therapy group were observed under confocal and there were EGFPs in pLEGFP-N1 group. No EGFPs were seen in blank control group. 70 Dacron patches plus 10 untransplanted Dacron patches were magnified by stereoscope (×160 fold) and electron microscope (×500 fold). We found that there were no thrombi on the surface of untransplanted and gene therapy group Dacron patches and there were many thrombi on the surface of control groups Dacron patches. Exogenous tPA bands were found in the inferior caval veins of gene therapy group through Western blot and no exogenous tPA bands in the veins of control groups. According to the plasma plates, big areas of thrombolysis were found in the plasma plates drop by substances from gene therapy group veins and this indicated strong thrombolysis. On the contrary, there were no thrombolysis in control groups. According to the activity of lumbrukinase, we counted that tPA activities from gene therapy group was 529.62±9.05u/g and 537.50±12.45u/g respectively on 2nd and 75th day after operation. The activities of tPA there were no significant variation between 2nd and 75th (P>0.05). The tPA contents of inferior caval veins from 6 sub-units were (737.64±13.19),(29.88±5.61),(28.7±5.49),(742.87±10.56),(32.03±6.26),(31.34±5.63) ng/g corresponding to A₁,B₁,C₁,A₂,B₂,C₂. Contents of tPA in gene therapy were obviously higher than that in control groups (P<0.01). tPA contents there were no significant difference between 2nd and 75th day in gene therapy group (P>0.05).Conclusion pLEGFP-N1-tPA locally transferring can effectively prevent thrombosis on the surface of exogenous transplant. This provide a deeply basis for the study and application of tPA gene valve.