| Objective:To establish the model that cervical carcinoma HCE1/MCS invade live HUVEC (HCE1/MCS invasion model) . To investigate the relationship between the expression of MMP-9 in the HCE1/MCS and cervical carcinoma infiltration .To probe the effect of MMPs inhibitor GM6001 on the model that HCE1/MCS invade live HUVEC. Methods:1,Establish the HCE1/MCS invasion model(1): Using liquid overlay technique and rotating culture technique to get the HCE1/MCS.(2): Culture live HUVEC.(3): HCE1/MCS cocultured with HUVEC: HUVEC at 70,000 cells/well added to 48-well plates and allowed to grow to confluence for 48h. HCE1/MCS were resuspended in complete media to obtain approximately 5-10 spheroids/ml, and 1ml of the suspension was added to each well atop the HUVEC, and then were incubated in a 5 % CO2 humidified incubator at 37℃for 7 days. On each day 1ml of complete media was superseded.2,The expression of MMP-9 in HCE1 monolayer cells (HCE1/MC),HCE1/MCS and HUVEC were detected by immunohistochemistry. 3,The HCE1/MCS invasion model were treated with MMPs inhibitor GM6001:(1): Groups:①Control group: Cells were treated with the same concentration of DMSO②GM6001 2.5uM③GM6001 12.5uM.(2): The effect of MMPs inhibitor GM6001 on HCE1/MCS invasion model: HCE1/MCS and HUVEC cocultured at 37℃for 1 hour, and then added GM6001 to each well. On each day 1ml of complete media and the same concentration of GM6001 was superseded. At time-point of 0, 1,4 and 7days from the initial plating, HCE1/MCS were rephotographed. The area of the invasive MCS were determined using Motic Med 6. 0 to analyze the inhibitory action of GM6001.(3): The expression of MMP-9 in HCE1/MCS invasion model(4 days) were detected by immunohistochemistry.Results:1,Establish the HCE1/MCS invasion model(1): HCE1/MCS: The growth of HCE1 monolayer cells were in good condition, after 24h in rotating culture,HCEl cells began to aggregated loosely. With the culture time extending, the spheroids became more and more compact and larger in size.(2): HUVEC: The growth of HUVEC was in good condition, adhence to irregular diagonal corniform looking like salbstone.(3): HCE1/MCS invasion model: After cocultured for 1h, HCE1/MCS adhere to HUVEC tightly. With the time prolong,HCE1/MCS disaggregated obviously, dispersed and infiltrated inHUVEC rapidly, while HUVEC retracted manifestly.2,The expression of MMP-9 in HCE1/MCS was significantly higherthan that of HCE1/MC(p<0.05).The expression of MMP-9 in HUVECwas negative.3,The HCE1/MCS invasion model were treated with MMPs inhibitorGM6001(1): Group GM6001 2.5uM: Compared with control group, the disaggregating of HCE1/MCS weakened, infiltrating area reduced and the retraction of HUVEC degraded.GM6001 inhibited HCE1/MCS invasion by 26.09%(p<0.05).(2): Group GM6001 12.5uM: Compared with control group,HCE1/MCS had not yet disaggregated. The infiltrating area reducedobviously and the retraction of HUVEC was almost not observed.GM6001 inhibited HCE1/MCS invasion by 92.95%(p<0.05).(3): The expression of MMP-9 in HCE1/MCS invasion model (4 days):The expression of MMP-9 in GM6001 2.5uM experiment group was lower than that of control group and there was no significantly difference between the two groups(p>0.05). The expression of MMP-9 in GM6001 12.5uM experiment group was obviously lower than that of control group (p<0.05)Conclusions:1. Established HCE1/MCS invasion model successfully. 2. The enhancement of HCE1/MCS infiltration maybe relate to the up-regulation of MMP-9 expression in HCE1/MCS3. GM6001 can partial block the HCE1/MCS invasion effect to HUVEC and down regulation the expression of MMP-9 in HCE1/MCS.
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