Effects And Underlying Mechanisms Of The Prescription Of Jiajian Zhujing On The Expression Of VEGF Of ARPE-19 Cells Induced By Transgenic Akt Gene

Objective1. To observe the effects of Jiajian Zhujing prescription on the proliferation of ARPE-19 cells induced by transgenic Akt gene.2.To explore the effects of Jiajian Zhujing prescription on the content of VEGF in cells induced by transgenic Akt gene cultivate supernatant, and to observe the effects of the prescription on the expression of Ak^ mTOR and HIF-la in model cells, demonstrate the possible mechanism of the prescription inhibit the formation of CNV.Methods1. To culture good ARPE-19 cells in vitro, and to divide into 4 groups, including normal serum group?blank serum group?medicated serum group and Conbercept group, and set up different observe time and different observe concentration.2. The vitality of every group cells cultured in different concentration were measured using CCK-8.3.Transducing complexes of pcDNA3.1-AKT plasimid to the ARPE-19 cells through According to Lipofectamine 2000 transduction, to establish cell model of over expression of AKT gene.4. The protein expression of AKT in model cells were analyzed using Western blotting and immunofluorescence, to demonstrate if it was success.5. To culture model ARPE-19 cells after tansfection with Akt gene(the same below) in vitro, and to divide into 5 groups, including normal serum group= model-normal serum group?model-blank serum group?model-medicated serum group and model-Conbercept group, and set up different observe time and different observe concentration.6. The vitality of every model cells group cultured in different concentration were measured using CCK-8.7. The concentration of VEGF in every conditioned model cells medium were measured using ELISA.8. The gene expression levels of AKT?mTOR?HIF-1a and VEGF in every conditioned model cells were detected using real-time quantitative polymerase chain reaction (real-time PCR).9. The protein expression levels of AKT?mTOR?HIF-la and VEGF in every conditioned model cells were detected using Western blotting and immunofluorescence.Results1. Compared with normal control, the vitality of blank serum group?medicated serum group cultured in different serum concentration for 24 hours, the difference of the vitality of ARPE-19 cells have no statistical differences (P>0.05). Be cultured at the concentration of 20% or 40% serum for 48 and 72 hours, the vitality of ARPE-19 cells be decreased as compared to normal control(P<0.05), while which of the 2. 5%?5%?10% was not unchanged(P>0.05).To Conbercept group, it was harmful to ARPE-19 cells at the concentration of more than 20?g/ml, there were statistically significant differences which compared these groups to normal control group(P< 0.05), and the concentration of 20?g/ml was safe (P>0.05). So we had the serum of 2.5%?5%?10%, and the 20?g/ml Conbercept used to investigation.2. Result of Western blot assay confirmed that:the Akt protein level of transgenic model cells was higher than normal control group (P<0.05), which demonstrate the model cell was success.3. Result of CCK-8 measure confirmed that:the proliferation of every model cells group cultured in different concentration is higher than normal control group for 24h and 48h (P<0.05). There was no different between blank serum group and normal (P>0.05), while the concentration of 5% and 10% model-medicated serum group, and the 10,20?g/ml model-conbercept group is lower than model cells group (P< 0.05). When they were cultured for 72h, the proliferation of model cells was about equal to normal control group (P>0.05),which indicated that it was decreasing with the time longer. At last, we started investigation in 24h and 48h, which chose the serum concentration were 5% and 10%, while Conbercept concentration for 10,20?g/ml.4. Result of ELISA assess shown that:the levels of VEGF to be significantly increased in model cells(P<0.05), which show that the over expression of Akt can promote the proliferation of VEGF expression; the blank serum group and model group compared, there was no statistically difference(P>0.05); the model-medicated serum group and model-Conbercept group compared to normal control group, there was statistical differences (P<0.05), which show they can restrain the production of VEGF after over expression of Akt. The expression of VEGF in 10% medicated serum less than 5%,10% group (P<0.05),20?g/ml Conbercept group than 10?g/ml as well. We take the best concentration 10% medicated serum group,20 ug/ml Conbercept in further experiment as treatment and positive control group respectively.5.Result of RT-PCR assess shown that:to culture model group cells normally for 24h and 48h, the gene expression levels of Akt, mTor, and VEGF were significantly higher than normal group (P< 0.05), explain that their expression were promoted after induced by transgenic Akt gene. About the gene expression levels of Akt, mTor, and VEGF, it has no statistical significance between blank serum and model group (P> 0.05). Medicated serum and Conbercept group were lower than model group (P< 0.05), which descript they can inhibit the gene expression levels of Akt, mTor, and VEGF after over expression of Akt; but HIF-la was unchanged(P>0.05).6. Result of Western Blot assess shown that:the result change of protein expression levels of Akt, mTor, and VEGF were same to RT-PCR assess, including HIF-1a protein.Conclusion1.Akt gene transfection can promote ARPE-19 cells proliferation, as well the protein and gene expression levels.2.Jiajian Zhujing prescription medicated serum can inhibit the proliferation of model cells, and the protein, gene expression levels of AK?mTOR?VEGF.3Jiajian Zhujing prescription medicated serum may restrain activity of AKT, regulate AKT/mTOR/VEGF pathway, to inhibit the expression of VEGF, thus inhibit or delay the formation of CNV.