| Objectives 1. To establish the muticellular resistance (MCR) model with cervical cancer cells.2.To explore the effect of CD147 on MCR of cervical cancer cells.3.Using CD147 monoclonal antibody(mAb) to block CD147 in order to observate its effects on reversing MCR and P-Glycoprotein(P-gp) expression.Methods1.To culture tumor spheroids of human cervical cancer line(HCE1) using liquid overlay and totating technique,monolayer cells using routine procedure. We observed the morphologic changes of cells with light microscope; Observation CD147 monoclonal antibody'role in HCE1 multicellular spheroid morphology change.2. To inhibit the activity of CD147 by different concentration of CD147mAb,then detection the inhibition of proliferation with WST-1 on both HCE1 monolayer cultured cells(HCE1/MC) and HCE1 muticellular spheroid(HCE1/MCS) before and after blocking the CD147. The 50% inhibiting concentration(IC50) of spheroids or monolayer cells for paclitaxel(TAX) is determined and the index of MCR(MCRI)is calculated. Then mapping the concentration-inhibition curve.Analysing the role of CD147 in MCR and its effect of reversing MCR.3.Four groups were measured(the control group, CD147mAb group, TAX group, TAX+CD147mAb group), the cell cycle and apoptotic rate of four groups are detected by flow cytometry machine.4.The high expression of CD 147 and P-gp in HCE1/MC or HCE1/MCS were detected by immunohistochemistry.Results1. we observed the growth of HCE1 monolayer cells were in a good condation, after 24h in rotating culture, cells began to aggregated into multicellular spheroids, and becoming more and more compact and larger in size relay to culture time;Monolayer with CD147mAb:loose connections between cells and can not grow into a film, control group: with close connections between the cells, into a film growth; Multicellular spheroid with CD147mAb group:can not form multicellular spheroid, or cells together, but loose structure form while control group with Stable structured.2.The inhibition rate of HCE1/MC and HCE1/MCS cells:in the control group, the value of IC50 for paclitaxel were 28.27±1.45μg/ml vs 64.7±1.67μg/ml (P＜0.05), MCRI is 2.29. When the activity of CD 147 in spheroid cells is inhibited by CD 147 mAb with different concentration(5,10,20μg/ml),CD 147 was down-regulated, paclitaxel can easily induce cells to apoptosis,the value of IC50 were 40.31±3.73μg/ml,32.43±1.56μg/ml,30.69±1.01μg/ml, MCRI were 1.48,1.20,1.18,compared with the control group(P<0.05).When in low concentration,the inhibition rate is dose-dependent,5μg/ml group vs 10μg/ml group(P<0.05),but in high concentration,it is non-dose-dependent, 10ug/ml group vs 20μg/ml group(P>0.05);The CD147mAb do no effect for monolayer cells,CD147 mAb groups vs the control group(P>0.05).3. Spheroid cells accumulate in the G1 phase of cell cycle(73.00%±2.65%).The apoptotic rate of spheroid cells is about 2.39%±0.42%, monolayer cells are adherent to the bottom of flask, cells are actively dividing and the rate of S phase is 42.20%±0.20%.The apoptotic rate of cells rises to 6.02%±0.20%;When treated with paclitaxel,both MC and MCS were proliferation inhibited,the apoptotic rate were:33.97%±1.97%vs 17.40%±0.53%(P<0.05),G2/M phase were 21.03%±1.05%,14.60%±0.46%,compared with the control groups (P<0.05); When treated with CD147mAb,the apoptotic rate of MCS was:13.77%±0.59%vs the control group(P<0.05),G1/G0 phase: 81.40%±2.16%vs the control group (P<0.05),S phase:12.67%±1.81% vs the control group (P＜0.05),G2/M phase:13.77%±0.59%vs the control group(P>0.05); When treated with TAX+CD147mAb,compared with the TAX group,the effect is like the above.CD147mAb do no effect to monolayer cells.4.The high expression rate of CD147 in both HCE1/MC and HCE1/MCS were:Increased expression of CD 147 and P-gp were observed in MCS compared with the monolayer cells,the high expression were:in control group,CD147 were 53.33% and73.33%(P<0.05); p-gp were 0 and 46.67%(P<0.05); Treated with palitaxel resulted in a remarkable overexpression of CD147 and P-gp in MCS, the high expression of CD147 and P-gp in TAX group were 93.33% and 73.33%,compared with the control group (P<0.05).while monolayer cells fail to show similar response(P>0.05).TAX group,60% and 93.33% (P<0.05); CD147 mAb group,0 and 3.33%(P>0.05);CD147mAb can inhibit the expression of both CD 147 and P-gp in all groups.Conclusion1.Paclitaxel resistance of HCE1/MCS model was established successfully.2.CD147 plays an important role in MCR of cervical cancer.Inhibition of CD 147 activity can synergistically reverse the multicellular drug resistance in cervical cancer.3.The MCR of cervical cancer caused by CD 147 is related to P-gp. |
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