Prokaryotic expression and purification of sheep granulocyte colony stimulating factor for granulosa cell culture

Abstract:

In order to clarify the regulation of granule cell stimulating factor (GCSF) on granulosa cells, we studied the effect of GCSF on proliferation and apoptosis of in vitro cultured granulosa cells for research on GCSF used in sheep reproduction and breeding. Sheep GCSF protein was prokaryotic expressed and purified. Its bio-activity was measured with M-NSF60 cells. The purified GCSF was added in cell culture medium in experiment groups with non-added as control. Alarmarblue was used to measure cell proliferation, and flow cytometry was used to detect cell cycles and apoptosis. Sheep GCSF could be expressed and purified. Cell activity increased with GCSF concentration from 0.06 to 600 ng/mL at 24 h and 48 h. Cell cycles were significantly different between experiment and control groups at 24 h. Cell ratio of S was significantly reduced (P<0.05) and G2/M phase significantly increased (P<0.05). The apoptosis ratio of experiment group was significantly reduced (P<0.05) at 48 h. In conclusion, GCSF could enhance cell proliferation, inhibit apoptosis, and regulate cell cycles on in vitro cultured sheep granulosa cells.