Construction Of PEGFP-PTEN Recombinant Eukaryotic Expression Plasmid And Expression On Human Glioma Cell Line

Objective: To provide basis for gene therapy scientifically. Firstly, a recombinant eukaryotic expression plasmid contained PTEN gene fused with EGFP (enhanced green fluorescence protein) gene was constructed, then detect the expression of the recombinant vector on human glioma cells. Secondly, the biological effects of the expression of PTEN gene on these malignant glioma cell line were investigated.Methods: (1) PTEN gene which abstracted from glioma cells U251, SHG-44 and SWO-38 were amplified by RT-PCR and sequenced. (2) The normal human PTENcDNA gene was amplified and inserted into pEGFP-Nl that was selected by T-A subclone. The recombinant expression vector was obtained. After the recombinant plasmids were transfected into the glioma cell lines with abnormal PTEN gene by cation polymex, expression of fusion protein was tested. (3) The stably transfected cells were selected by G418 and amplified. Light microscope and growth curve were used to measure the effects of PTEN expression on cell morphology and proliferation. Expressions of related protein products were detected by Western blot and immunohistochemistry staining.Results: (1) Point mutation, deletion and wide-type were observed respectively in the amplified products of PTEN mRNA for U251, SHG-44 and SWO-38. (2) The positive recombinant was sequenced and demonstrated to have the same sequence as that of PTEN gene in Genbank. It was proved that the eukaryotic expression vector pEGFP-PTEN have been constructed successfully. Fusion protein expression was detected by the recombinant vector transfected. Expression of EGFP protein was found by fluorescence microscopy and flow cytometry and expression of exogenous PTEN protein was found immunohistochemically in transient transfected glioma cell. (3) Cells proliferation was inhibited obviously in stably transfected cells during the 7-day period. The cell counts of stably transfected U251 and SHG-44 on the 7th day are 39.1 % and 27.8 % of untransfected cell numbers. Expression of PTEN protein and GFAP (glial fibrillary acidic protein) both increased. The different effects on cell morphology were also recognized. The stably transfected U251 cell differentiated toward astrocyte, but SHG-44 cell did not.Conclusion: The construction eukaryotic expression vector pEGFP-PTEN will lay a basis for carrying out further studies on the function of PTEN gene. It could induce differentiation differently on glioma cells which restore the expression of wild- type PTEN, and the different differentiation effects might associated with tumor Heterogeneity.