A Study In The Effects Of Recombinative Adeno-associated Virus Expressing Repressor Of GATA-3 On Protection Asthma

Objective: l.To establish antigen-specific T helper 1 and T helper 2 cell lines,and analysis the cytokine profiles that they secret.2.To clone the gene of murine repressor of GATA-3 and construct the recombinative adeno-associated virus vector named pAAV-ROG harboring ROG,then product recombinative adeno-associted virus(rAAV-ROG),to investigate the effect of rAAV-ROG on protecting the mouse from the asthma.Method: Lymph node T cells from BALB/c mice after being immunized with OVA for 10 to 14 days were repeatedly stimulated with OVA and spleenic cells from BALB/c mice to establish OVA-specific T cell line. Limited dilution method was applied to abtained the single cell relatively.After that,T cells were stimulated with IL-2 and IFN Y (Th1 skewing condition) or IL-2 and IL-4(Th2 skewing condition) for 10 to 14 days repeatedly.ELISPOT was applied to measure the cytokines profiles of these cells and determined which subset they belonged to.T cells clone,Thl or Th2 cells clone were obtained by limiting dilution technique.Two primers were designed according to the sequence of ROG that genebank publiced.Total RNA was extracted from cultured CTLL-2 cells.The gene sequence which codes ROG were amplified with one-step RT-PCR.After sequencing,the fragment was inserted into AAV vector(pAAV-MCS) ultimatly to obtain the recombinative pAAV-ROG that harboring the ROG;then Hela cells was transfected with pAAV-ROG and pAAV-rhGFP was the control plasmid to identify the ROG gene was expressed or not.Several plasmids such as pAAV-ROG,pAAV-RC,pHelper and pAAV-rhGFP,etc were extracted and adjusted them to the concentration of 1 μg/ml.AAV-293 cells were cultured in DMEM with high glucose and passaged till about 50% of cofluence.When confluented up to 70~80%,AAV-293 cells werre contransfected with pAAV-ROG combinated pHelper and pAAV-RC by calcium phosphate methods.All transfection samples were divided into 6 groups:A,B,C,D,E,and F.Group A was added no any other plasmids, group B was transfected by all three plasmids named pAAV-ROG,pHelper,pAAV-RC,all of them being 10 μg respectively. In group C and D pAAV-ROG was replaced by pAAV-rhGFP and pAAV-LacZ to perform the transfection.In group E and F,pHelper or pAAV-RC was disregarded and the other contents were the same as the group B.A11 cultures were replaced fresh medium after transfection six hours.The change of the mediumand cells was observed under the light microscope.Seventy-two hours later,AAV-293 cells and media were collected respectively,then placed in 37°C water bath and dry ice alternatively for three times and collected cellular debris by centrifugation at 10,000 xg for 10 minutes at room temperature.The supernatant(primary virus stock) was transferred to another fresh tube.Viral stocks were stored at -80°C.More advanced condensation could be adopted alcohol methods.The rAAV-ROG, rAAV-rhGFP titres were assessed by measuring the rAAV-LacZ with dot blotting.The asthma model of Balb/c mice were constructed through ip injection ovalbumin,in 1,8,15 days,with a dose of 10 u g OVA/mouse/times.Triggering was performed in 26,27,28 days, with a dose of 1% 0VA.In the forteeth day,the mice were given 50 u 1 rAAV-ROG intranasally. All animals were anesthesiaed and performed bronchoalveolar lavage then sacrificed,their lungs were stained with HE and assessed by immunohistochemistry staining.The cells in BALF were counted and conducted evaluating statistically.Results:Five T subset cell clones ,with Thl and Th2 cell like of each subset were obtaine.H-2 limited analysis implied that these cells could have high proliferation index only spleen cells were imployed as antigen presenting cells.Antigen-specified analysis indicated that these cells could proliferate only under OVA was imployed as antigen.Through different inducing factors the cells could express different cytokines by ELISPOT.The ROG cDNA was amplified by RT-PCR from total RNA extracted from cultured CTLL-2 cells.The resultant fragment was cloned into pAAV confirmed by restriction digestion and sequencing. After transfection, some of the cells will round up and detach from the plate, and can be seen floating in the medium.The medium changed its colour from red to yellow for group B,C,D.Especially to the group C,under the fluencent microscope,green fluencent light could be observed after transfecting 10 hours.Under the microscope photographs AAV-producing versus non-AAV-producing AAV-293 cells show distinct profile. High titres of recombinative virus was produced through condensing them several times.Mice model were reproducted by OVA sensitization and stimulating according the rutine.Cells counted in BALF and the lungs' HE and immunohistochemistry pictures showed that the rAAV-ROG had the effect of preventing mouse from OVA-induced asthma.ConcIusion:OVA-specific Thl/Th2 cells were obtained and cultured in vitro.Recombinated adeno-associted virus that expressed the mouse repressor of GATA-3 gene could have the effect on protect the mouse from the asthma.