| Purpose:In this study,HUC-MSCs and tongue squamous cell carcinoma(TSCC)cells were co-cultured,CCK-8 kit was used to measure cell proliferation,and Western blot was used to investigate the effect of HUC-MSCs on the growth of tongue squamous cell carcinoma Cal-27,so as to provide a new idea for the treatment of oral TSCC.Methods:1.Transwell cell co-cultureThe HUC-MSCs in logarithmic growth phase and the tongue squamous cell carcinoma Cal-27 were prepared cell suspension and counted,and the cell concentration was adjusted to 6×10~5cells/ml.A tannswell chamber with a 0.4um pore size polycarbonate membrane was selected and placed in a 6-well culture plate and Cal-27 cells were placed in the upper chamber.After culturing for 72 hours,the process were stopped,fixed in methanol and stained with crystal violet.The number of cells was observed by photographing.2.Preparation of conditioned mediumWhen HUC-MSCs cells grew to 80%or more in length,they were cultured in thesame culture conditions with the addition of fetal bovine serum-free MSCM medium.After 24 hours,the upper medium was aspirated into a sterile centrifuge tube with a sterile pipette and centrifuged.The supernatant was filtered through a 0.22?m sterile filter and stored at-80°C until use.3.CCK-8 cell proliferation assayCal-27 cells were co-cultured with different concentrations of HUC-MSCs conditioned medium for 72 hours.CCK-8 was used to detect the effect of HUC-MSCs supernatant on the proliferation of Cal-27 cells.5×10~3Cal-27 cells were seeded in 96-well plates(100?l/well).After the cells were adhered,5%,10%,20%,40%,and 60%HUC-MSCs conditioned media were added.The non-conditioned medium of Cal-27 cells was used as a control group.After co-culture for 72 hours,absorbance(OD)at a wavelength of 450 nm was measured with a microplate reader.4.Western blot methodThe Cal-27 cells were seeded in 6-well plates.When cells were over 80%confluent,the experimental group was replaced with 60%conditioned medium to culture Cal-27cells.The control group was still DMEM high-glucose medium at 37°C.After culturing in5%CO~2 saturated humidity incubator for 72 hours,the total protein of Cal-27 cells was extracted,and the expression differences of bcl-2,PARP-1,?-catenin,and c-Myc in the two groups were detected by Western blot.The final statistical analysis was performed.Results:1.It was found through experiments that the staining of crystal violet in the co-culture group was significantly less than the number of Cal-27 cells in the control group.2.After adding 5%,10%,20%,40%,60%HUC-MSCs conditioned medium,the inhibitory rates of HUC-MSCs to Cal-27 cells were 5.35%±1.76%,10.99%±2.91%,17.20%±1.75,24.52%±2.95%,40.70%±1.01%,compared with the control group,the difference was statistically significant(P<0.001).3.60%HUC-MSCs conditioned medium was used to treat Cal-27 cells for 72 hours,the expression of apoptosis-related protein PARP-1 was significantly up-regulated,and the expression of anti-apoptotic marker protein Bcl-2 was significantly inhibited.?-catenin,c-Myc expression was significantly down-regulated,with extremely significant differences(P<0.001).Conclusions:1.HUC-MSCs inhibited the growth of Cal-27 in tongue squamous cell carcinoma.It was presumed that HUC-MSCs could secrete certain factors to affect the growth of Cal-27.2.HUC-MSCs conditioned media can inhibit the proliferation of tongue squamous cell carcinoma Cal-27.3.The expression of PARP-1 and Bcl-2 apoptotic protein in tongue squamous cell carcinoma Cal-27 was significant by HUC-MSCs conditioned medium,indicating that HUC-MSCs conditioned medium can promote the apoptosis of tongue squamous cell carcinoma Cal-27.Simultaneously,the expression of?-catenin and c-Myc were significantly down-regulated.It was speculated that the conditioned medium of HUC-MSCs might affect the Wnt/?-catenin signaling pathway in tongue squamous cell carcinoma cells and induce apoptosis of Cal-27. |
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