Proteomic Analysis Of Two Strains Of MEF Cells With PTEN Deletion

Pten is a tumor suppressor gene with phosphatase activity, and has been found in various cancers. It is estimated that the overall mutation frequency of Pten in human cancer can compete with p53. Pten is one of the hot spots in studied tumor genes recently.One of the most important methods in further pursue with gene is culturing Pten conditional knockout mouse. There are four labarories which have done this successfully. The mouse with Pten conditional knockout embryonic fibroblast was prepared in Hong Wong's labarory: MEF1/Pten^-/- and MEF2/Pten^-/-, from primarily cultured MEFIPten" cells with immortality. Our research was based on this mouse model.To investigate the unknown machenism regulated by Pten .firstly, we screened the proteins by using Proteomic technology. The development of 2-DE, MS and bioinformatics allow us to assay the proteins differential expression between MEF and MEF/Pten^-/- cell lines. In the result, Cu/Zn-SOD, PeroxiredoxinV-VI, Cofilin 1, GDI2, LMW-PTP, Tagln 2 et al , have been found. Subsequently ,to further identify these proteins with molecular biology techniques and some biologic activity with them were detected also. Additionally, in this reseach, we using MEF2/Pten^' cell line as the model, refered to MEF1/Pten^-/-, final results as following:1. After comparing the gene express profiles between MEF2 and MEF2/Pten^-/-,The Northern and Western blot results show that MEF2/Pten^-/- cells has more Cu/Zn-SOD than it's normal control ,and also it's enzyme activity has strengthened. Using flow cytometry analysis , more O₂^- in MEF2/Pten^-/- cells was detected. from these effects to hint that oxidative stress has happened in Pten^-/- cells, and the overexpresion of Cu/Zn-SOD may related to O₂^- and AKT signal transduction path.2. The Northern and Western blot results appear that MEF2/Pten^-/- have more PRXs than their normal cell line, but interestingly only PrxVI underexpressed in this experiment. Using flow cytometry analysis , less H₂O₂ in MEF2/Pten^-/- cell was detected.But to be surprised, the effect is inverted after incubating with 0.5mM H₂O₂ . All of these phenomena reveal that PRXs may play a vital role in scavenging H₂O₂ in Pten^-/- cells. And maybe exist a minus-regulation between SOD and PRX VI when Pten is null in cells.3. After deletion of Pten , Cofilin 1 manifest up-regulation and GDI2 down-regulation in cells. All of these occurrences are in line with malignant tumor phenotype.4. Other relative proteins : such as LMW-PTP, Tagln2 ,being down-regulated with mRNAs inMEF2/Pten^' cells.These findings indicate that in MEF2/Pten^<sup> cells oxidate stress has happened ,oxidate equilibrium has been demolished and accommodating adjustment has appeared both Cu/Zn-SOD and PRXs, negatively regulation to cell growth, proliferation attenuated, migration ability potentialized. Above all ,our research may contribute to deeply understand with Pten gene.Postgraduate student: Yu changhong (Medical Genetics) Directed by: Wang peilin professorLi gang research worker...