The Effects Of Progastrin、Gastrin、Annexin2on Biological Behaviour Of CD44+Gastric Cancer Cell

Objective:(1) To identify whether the CD44+cell are successfully sorted, and study the characteristic of tumor stem cell.(2) To investigate the expression of progastrin, annexin2, gastrin in CD44+cell, BGC-823cell, CD44-cell.(3) To study the effect of progastrin, annexin2, gastrin on the proliferation, invasion in CD44+cell and BGC-823cell.Methods:(1) In order to identify whether the CD44+cells are successfully sorted,we employing flow cytometry and cell immunofluorescence to detect the expression of CD44in CD44+cells. Flow cytometry-cycle and serum-free culture methods study CD44+cells possess the characteristic of stem cells.(2) The expression of progastrin、gastrin、annexin2in CD44+cells, BGC-823cells and CD44-cells was detected by flow cytometry, cell immunofluorescence and cell immunohistochemistry.(3) The effect of progastrin, gastrin, annexin2on proliferation of CD44+cell and BGC-823cell were detect by MTT assay, Boyden chamber was employed to detect the effect of them on invasion of CD44+cell and BGC-823cell.Results:(1) The expression of CD44in CD44+cells (90.20±2.24) is significantly higher than BGC-823cells (9.57±2.35)(P<0.01). The cell percentage of G0/G1phase of CD44+cells (71.1±5.6%) were obviously higher than BGC-823cells (47.7±3.5%)(P<0.05).The CD44+cells in serum free cultured for7days could form a circular clone ball, however CD44-cells cultured in serum free medium7days could not form a circular clone ball;(2) The expression of progastrin (55.29±3.84), gastrin (55.08±4.10) and annexin2(48.33±1.45) in CD44+cells were significantly higher than the expression of them in BGC-823cells and CD44-cell (P<0.01).(3)1mg/ml and2mg/ml progastrin mAb,0.5mg/ml,1mg/ml,2mg/ml gastrin mAb,1mg/ml and2mg/ml annexin2mAb could inhibit the proliferation of BGC-823cell, there was statistical significance compared with control group,(P<0.05orP<0.01).(4)1mg/ml,2mg/ml progastrin mAb and0.5mg/ml,1mg/ml,2mg/ml annexin2mAb could inhibit the proliferation of CD44+cell, there was statistical significance compared with control group,(P<0.05orP<0.01); but gastrin mAb could not,(P>0.05).(5) The number of invasive BGC-823cells,which were treated with progastrin mAb, gastrin mAb, annexin2mAb, respectively were40.39±10.61、43.17±7.63、42.34±5.66, there was statistical significance compared with control group,(P<0.05).(6) After treatment of progastrin mAb, gastrin mAb, annexin2mAb, the number of invasive CD44+cells respectively were70.15±4.77、76.42±9.45, there was statistical significance compared with control group,(P<0.05); the group of gastrin mAb can not inhibite the invasive of CD44+cell,(P>0.05).Conclusion:(1) The result of flow cytometry-cycle and serum-free culture,which suggest CD44+cell possess the quality of tumor stem cell.(2) CD44+cells widely express progastrin, gastrin, annexin2, which suggest them have the relation with gastric cancer stem cells.(3) Progastrin mAb, gastrin mAb and annexin2mAb can inhibit the proliferation and invasion of BGC-823cell, which suggest that progastrin, gastrin, annexin2promote the proliferation and invasion of BGC-823cell.(4) Progastrin mAb and annexin2mAb can inhibit the proliferation and invasion of CD44+cell, which suggest that progastrin and annexin2promote the proliferation and invasion of CD44+cell.