| Objectives Esophageal carcinoma（EC）EC9706cells are incubated in vitro. Thepurpose of the study is to observe the inhibition/proliferation mechanism of serum in ECpatients with blood stasis syndrome（BSS）on EC9706cells, through analyzing theinfluence of serum on cells cycle and investigating the influence of serum on PI3K/AKTSignal Pathway in EC9706cells. Molecular mechanism of blood internal environment onthe occurrence of esophageal carcinoma and the pathogenesis of dysphagia of blood stasissyndrome is to be illustrated.Methods EC9706cells were inoculated on Φ100mm Petri dishes, with10%calfserum DMEM culture medium, the temperature was set at37℃, saturated humiditychamber incubation for5%CO2culture, cells were adherent cells24h,24h for starvation,offered with serum in EC patients with BSS（T group）, spleen-Qi deficiency syndrom（Q group）and healthy volunteers（group P）.6serum concentration gradients wereset, including31.25，62.5，125，250，500and1000μl/10ml. Cells in control group（C group） were incubated by total free medium. Proliferation was assayed by MTT.Cells morphology was observed with microscope and cell cycle was measured with FlowCyto Meter（FCM）. Associated proteins within PI3K/AKT signaling pathway weredetermined by western blot.Results1. Serum of EC patients with BSS stimulated EC9706cells proliferation.Serum concentration was positively correlated with cell proliferation rate and the fittingcurve equation was protracted with F test in SPSS. The T group of PI50=840μl/10ml, Qgroup of PI50=1180μl/10ml, P group, PI50=1243μl/10ml.2. Influence of serum of ECpatients with BSS on morphology of EC9706cells was as follows: compared with the Pgroup, T group and Q group cells proliferated sharply, body slants big, plump, cell gapnarrow; cells in culture medium convergence rate was high, there was a great differencein the cell number and morphology. Cells connection in group Q was not very closely,cells in T group connected into the network.3. Influence of serum in EC patients withBSS on cell cycle of EC9706cells: Table2shows that the T group had obvious effect on promoting cells proliferation. In S phase of cell cycle（proliferative）, the percentagewas （38.85±3.11）%. Compared with group C, group T cell percentage in S phasewas significant different.（P＜0.05）.4Influence of serum of EC patients with BSS onPI3K/AKT signaling pathway of EC9706cells: Western blot showed that EC9706cellswere treated by serum of EC patients with BSS, protein bands in PI3K/AKT signalingpathway increased predominantly, which indicated that serum of BSS stimulated proteinsoverexpression. The expression intensity of the order is T>Q> P> C．Conclusions1. Serum of patients with BSS stimulated the cell proliferation of EC9706cells and induced cells into S proliferation period. It has close relation to proteinsoverexpression in PI3K/AKT signaling pathway.2. Blood internal environment ofpatients with BSS dysphagia was important factor to the occurrence and pathogenesis ofesophageal cancer. |
