| Objective:Observe the expression level of protein with epithelial-mesenchymal transition in human colon cancer cells SW480 in vitro,which were treated by mesenechymal stem cells.Study the regulation effect of MSC on Wnt/β-catentin signaling pathway, and the expression level of target genes (MMP-7, COX-2,β-catentin). The goal is to established the influence and probable mechanism of MSC on human colon cancer cells metastasis,and to provide some theoretical evidence for MSC in clinical practice for colon cancer.Methods:Culture of MSC and SW480 cells in vitro. Collect nutriaaant solution of MSC (3-8 generation). Separate SW480 cells into control group and experimental group.Control group by DMEM nutrient, experimental group by different concentration nutrient solution of MSC (20%,40%,80%). 1 Inhibition effect of MSC on SW480 cells with different group was measured by MTT, screening the clear superiority time of antiproliferation.2 View the scratch changes for 48h by scratch experiment.3 Flow cytometry (FCM) was used to measure the intracellular E-cad and N-cad fluorescence intensity of SW480 cells after treatment with MSC (20%,40%,80%) for 48h.To screening the clear superiority concentration of inhibition effect combine scratch experiment and FCM.4 The expression level of downstream target genes (MMP-7,COX-2) was analyzed by FCM.5 Expression of MMP-7,COX-2,β-catentin mRNA in SW480 cells after treated without and with MSC (40%) for 48h were detected by RT-PCR.Results:1 The results analized by MTT showed that:Compared with the control group, proliferation have significant changes after SW480 cells were treated with different concentrations MSC for 24h, the antiproliferation was (47.0±8.3)%, (72.4±4.8)%, (78.3±4.7)% in different groups, there was statistically significant differences (P<0.01). And there was statistically significant differences compared within different group(P<0.01).After added MSC for 24h,48h,72h, the antiproliferation in SW480 cells increased from (47.0±8.3)% to (56.0±3.8)% in 20% group,(72.4±4.8)% to (91.0±5.1)% in 40% group,(78.3±4.7)% to (93.3±4.9)% in 80% group. The inhibition efficiency was stepping up as the time increasing.And there was statistically significant differences compared between 48h and 24h(P<0.01).But there was not statistically significant differences compared within 72h and 48h(P>0.05).MTT results:The remarkable result of inhibition effect at 48h.2 Scratch experiment assay results:Observe by microscope,after 48h, compared with the control group, experimental group SW480 cells was moving slowly. The scratch at control group was thinner than experimental group.The width of scratch:control group<20% group<40% group=80% group.There are no change in the group 40% and 80%.The result prompt: MSC has inhibition effect for moving of SW480 cells.3 FCM assay E-cad and N-cad:E-cad goes steadily up after added different concentration MSC treatment for 48h, and N-cad goes steadyily decline.Conform to the change when EMT inhibited. FI of E-cad increased from 1 to 1.148, N-cad decreased from 1 to 0.977,compared with the control group,there was significant differences (P<0.05).20% group was significant differences compared with other treated groups(P<0.05). But, there was no significant differences between 40% group and 80% group(P>0.05).The remarkable result of inhibition effect was 40% group.4 FCM assay MMP-7 and COX-2:MMP-7 decreased from 1 to 0.965±0.007 after added 40% concentration MSC for 48h,COX-2 decreased from 1 to 0.89±0.0035, compared with the control group, there was significant differences (P<0.05).5 The results analized by RT-PCR showed that:Wnt/β-catentin downstream target genes (MMP-7, COX-2) were down regulated after SW480 cells were treated with MSC for 48h.MMP-7 decreased from 1 to 0.842, COX-2 decreased from 0.558 to 0.165, andβ-catentin mRNA have significant changes,from 0.660 to 0.215. There was statistically significant differences compared to the control group(P<0.05).Conclusion:1 MSC could inhibit the moveing of human colon cancer cells SW480 in vitro;its inhibition effect on SW480 cells may be related to epithelial-mesenchymal transition inhibited.This finding provide the new therapeutic pathway for cancer.2 The mechanism of MSC on SW480 cells may be related to Wnt/β-catentin signaling pathway.3 Provide some theoretical evidence for MSC to treatment colon carcinoma in clinical practice. |
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