Functional study of Tob gene related to Glioma

Objective To study the function of Tob gene in human glioma cell line SHG-44 by Lipofectamine transfection so as to offer some clues for the further experimental therapy. Methods According to Tob's Genbank accession No. D38305, pTracer-EF/V5-His A expression vector with green fluorescent protein (GFP) and Zeocin resistance gene. The above plasmid was introduced into SHG-44 glioma cells by Lipofectamine and the cells were observed under an inverted microscope at 2 h, 4 h, 8 h, 16 h, 24 h, 36 h, 48 h of the different culture time points. The smears of the transfected and non-transfected cells of about 105 each taken at 48 h were prepared and photographed under fluorescent microscope and analyzed using GFAP immunohistochemical staining, external growth curve and flow cytometer. Results Restriction enzyme digestion and DNA sequencing analysis showed that the Tob gene has been cloned and transfected into SHG-44 cells successfully. Mixing 4 μg Lipofectamine and 2 μg DNA and selecting by 10 μg/mL Zeocin can get more than 90% transfection-positive cells. After transfection, SHG-44 cells with transfected Tob gene grew slowly and even got into stillness. GFAP positive rate in these cells are higher than ones with non-transfected Tob gene. The growth speed of the cells with transfected Tob gene got slowly, even to a growth arrest. Flow cytometry revealed that after cultured in vitro for 24 h the positive cells in G2/M period decreased remarkably in number and the ratio of apoptotic cells increased notably. Conclusion The growth of SHG-44 glioma cells was suppressed by Tob gene transfection, and the cells tended to differentiate into normal gliocytes and apoptosis. So it was confirmed that Tob gene is really a novel antiproliferative gene of glioma cells, worthy of further functional analysis and research on experimental gene therapy.