Establishment Of An Immortalized Mouse Brain Astrocyte Cell Line

Astrocyte, a kind of macroglial cell, plays important role in the progress of neuro development, normal physiology and pathologic courses. Astrocyte dysfunction can cause neuron degeneration or neurodegenerative diseases. Most of primer astrocyte in vitro for study is separated from brain tissue. However, the process is tedious and time-consuming, and sometimes some primer astrocyte showed different characteristics because of sample batches variations. Moreover, primer astrocyte multiplication capacity in vitro is only about ten generations. This limits its use for lots of research. So establishing a stable actrocyte line is valuable. In this study, stable mouse brain astrocyte line with good characteristics was constructed by mediating human telomerase reverse transcriptase gene into astrocyte genome. Following are the main results:(1) By trypsin digestion and low speed centrifugation, the primer astrocyte was acquired and further purified by continuously passage. The cells presented fusiformis or polygon, uniform morphology, exuberant multiplication, anchorage dependent and good refractivity, cell body extension and own protuberance. Staining the specific proteins of astrocyte GFAP with immunofluorescent method showed positive. (2)Over expression plasmid vector pCDH-CMV-hTERT-EF1-copGFP was constructed, then identified with PCR amplification method and double restriction enzyme digestion method. After packaging viral with 293T cells and collecting virus supernatant, titer reached 5.12x108 TU/ml-15.37×108 TU/mL. (3) After mediating human telomerase reverse transcriptase gene into the forth generation astrocyte genome by lentivirus, positive astrocyte was sorted according GFP mark protein by flow cytometry. The cells were named as hTERT-KMAS cells. The perfect clone cells were chosen, enlarged and passaged to 70th generation. hTERT protein expression levels in 55th passaged hTERT-KMAS cells were detected by immunofluorescence method. Results showed that hTERT presented positive in hTERT-KMAS cells.(3) Compared to primer astrocytes, hTERT-KMAS line showed faster growth rate, stronger exuberant multiplication, and the same karyotype (2n=40). There were no significant differences between hTERT-KMAS and H22 cells in the telomere length, but both longer than the primer astrocyte, which indicated that the expression of telomerase tended to be stable. Moreover, hTERT-KMAS could not form cell clone in soft agar, which indicated that it was still kept adherent growth and communication.In conclusion, we successfully established a stable mouse astrocyte line, which could afford enough materials for further astrocyte study.