Transfection Of SV40T Into Astrocytes From Rat Cerebral Cortex

Objective To culture and purify astrocytes from rat cerebral cortex to obtain an in vitro culture model of high purity astrocytes and observe their growth and the biological features, and then transfect the gene-SV40T into astrocytes to be immortalized for further study of the characteristics of astrocytes in different developing stages.Methods Cells from Wistar rat cerebral cortex disassociated by enzymatic digestion and mechanical methods were treated with differential attachment and cultured for 7 to 10 days, and then were purified in orbital shaker. The purified astrocytes were identified by GFAP-immunoreactivity, then the plasmid pSV3neo with immortalizing gene-SV40T was transfected into the astrocytes by lipofectamine, and the transfection was labelled with SV40T antigen immunofluorescence.1. Purification and morphological observation of astrocytes:The neonatal rat brain was taken and the cortex was made into suspension by aids of enzyme-digestion and mechanical dissociation. In the process of culture, fibroblasts were reduced with differential attachment and oligodentrocytes and microglial cells were eliminated with orbital shaker. The growth features of the cultured cells were observed and the astrocytes were identified with GFAP immunoreactivity. Cell density, length of processes and cell branches were tested stereologically in different stages to examine the growth of the cortical cells in the culture.2.Plasmid extraction and identification:The competent cell DH5a was transfected by plasmid-pSV3neo, cultured and shaked at 37℃for amplification. From the culture the plasmid-pSV3neo was extracted and biocatalysted to be identified and then amplified for further study.3. Plasmid transfection:In 24 well dish, for one or two days culture, when the purified astrocytes fused to 80% or 90%, the lipofectamine and the plasmid in their own gradients of 3.0,4.0,5.0,6.0μl/50μl and 0.4,0.8,1.2,1.6μg/50μl respectively were pooled into the culture, incubated for 48 hours, and then labelled with immunofluorescence and counted the SV40T positive astrocytes to screen the optimal cell density, concentration of lipofectamine and plasmid for transfection of the purified astrocytes with SV40T in large scale.4.Observation of transfected astrocytes:Under the inverted phase contrast microscope, changes in morphology and biological features of cells were examined during and after transfection, and differences of astrocytes before and after transfection were also observed. The expression of SV40T antigen and GFAP in the transfected astrocytes was tested with immunofluorescence.Results1. Morphological changes of cultured cells:Cortical cells began to adhere to the container in one hour after plantation, after 6 to 8 hours, most cortical cells attached to container, and took the ellipse, fusiform and irregular shapes, some cells had small protrusions already. As times went on, the number and volume of cells increased, there were more and longer branches. The cultured cells proliferated the fastest from the third to the fifth day, when the cell number and volume increased rapidly. Cultured for 7 to 10 days, the cultured cells fused and got demix with astrocytes underlying, on top of which were neurons, oligodendrocytes and microglial cells.2.Purification and identification of astrocytes:Treated with orbital shaker, and passaged, cells demonstrated identical shapes rich of protrusions and their processes interlaced as a network. Labelled with GFAP-immunocytochemistry, the purified cells with positive immunoreactivity were stainned brown in their endochylema and processes. The positive immunoreactivity cells, the astrocytes, were counted more than 98 percent of all cells.3.Identification of plasmid:The extracted plasmid was cut into four clips with molecular weight of 4.6,3.1,1.4,0.64 kbp matched the Marker, suggesting that the plasmid was just what we needed.4. Optimization experiment:When cells reached a confluence of 90% and incubated with lipofectamie 5.0μl/50μl and plasmid 1.2μg/50μl, the transfected cells showed more SV40T-positive immunofluorescence, indicating the optimal transfection.5. Observation of transfected astrocytes:When cells fused to about 90%, the mixture of lipofectamine and plasmid was added to the culture, and incubated for 5 hours, the cell body swelled, thus the cells became clearer in their appearance. Forty-eight hours later, some cells schizolysised into pieces, while the rest remained unchangeable. Stained with immunofluorescence, the transfected astrocytes showed red-GFAP fluorescent light in their endochylema and processes, and green-SV40T fluorescent light in their endochylema.Conclusions1.The cultured cells proliferated the fastest when they were cultured for 3 to 5 days, the cell number and volume increased rapidly and their processes interlaced as a network. When the cultured cells fused and got demix from the seventh to the tenth day, cells were treated with orbital shaker and passaged, we got astrocytes which accounted for more than 98 percent of all cells.2. The optimal transfection condition by optimization experiments was gained when cells grew by 90% fused, and incubated with lipofectamie 5.0μl/50μl and plasmid 1.2μg/50μl in 24 well flat bottom.3.Plasmid-pSV3neo was successfully transfected into purified astrocytes.