| Objective: To observe the effect on colon cancer immunotherapy by Leucine-rich repeat containing G-protein-coupled receptor 5(Lgr5)activated dendritic cells(DC)induce antigen-specific CD8~+ cytotoxic T lymphocytes(CTL)and offer help to colon cancer immunotherapy.Methods: After DC maturation induced by Lgr5(Lgr5-DC),surface molecules DC80 ? DC83 ? DC86 and HLA-DR were tested by FACS Calibur/Calibur flow cytometry.Interleukin(IL)-12,and IL-10 of these maturation DC were tested with ELISA experimentation.Subsequently,Lgr5 antigen specific CTL CD8~+(Lgr5-DC-CD8~+ CTL)was induced by Lgr5-DC.The killing effect of the Lgr5-DC-CD8~+ CTL on normal colonic epithelial cells CCD-18C0 and colon cancer cell HT29 was detected by the apoptosis rate with FACS Calibur/Calibur flow cytometry.And the ELISPOT test was carried out to detect the liberation of the IFN-? after the normal colonic epithelial cells(CCD-18C0)and colon cancer cell HT 29 were incubated with Lgr5-DC-CD8~+ CTL.After Lgr5-DC-CD8~+ CTL treatment,tumor volume of colon cancer tumor bearing BALB/C-nu/nu mice was detected.The immunohistochemistry of the cell proliferation associated antigen(Ki-67)and micro-vessel density(MVD)associated with CD31 were performed with the euthanized mice.Meanwhile,the changes of tumor tissue after treatment were observed by HE staining.Results: Compare with PBS,Lgr5 protein stimulation can significantly increase surface markers DC80(42.7±2.8%)?DC83(44.0±2.8%)?DC86(45.6±2.9%)and HLA-DR(44.2±3.0%)levels,and up to 3.29,3.06,2.90 and 6.93 times(P<0.05).The ELISA results indicate that the Lgr5 stimulation significantly stimulated the release of IL-12 from the DC cell(373.5±8.9 pg/ml)and significantly reduced the secretion of IL-10 from the DC cell(231.6±6.7 pg/ml)(P<0.05).The results of the ELISPOT demonstrated that the amount of INF-? was approximately equal after the CCD-18C0 was incubated with the DC-CD8~+ CTL and Lgr5-DC-CD8~+ CTL(42.3 ±2.1 Spots/1×105 cells and 44.1±1.8 Spots/1×105 cells);However,the amount of INF-? after the HT29 cells was incubated with Lgr5-DC-CD8~+ CTL was significantly increased(182.5±5.2 Spots/1×105 cells),the INF-? amount of DC-CD8~+ CTL group was 44.0±1.4 Spots/1 × 105 cells(P < 0.05)? The apoptotic rate of the CCD-18C0 in Lgr5-DC-CD8~+ CTL group and DC-CD8~+ CTL group was 11.8±0.8% and 12.0±1.0%(P>0.05),respectively;However,the apoptotic rate of the HT29 in Lgr5-DC-CD8~+ CTL group and DC-CD8~+ CTL group was 54.6%±1.7 and 12.4±0.9%(P < 0.05),respectively.DC-CD8~+ CTL and Lgr5-DC-CD8~+ CTL both resulted in a small amount of CCD-18C0 cell killing(P>0.05),but the killing rate of Lgr5-DC-CD8~+ CTL on HT29 cells was 4.40 times as much as that of DC-CD8~+ CTL.In the immunohistochemical trials of related mechanism,a small number of Ki-67 positive cells was observed in the Lgr5-DC-CD8~+ CTL group(21.7 ± 7.5%)compared to DC-CD8~+ CTL group(40.6 ± 4.4%,p < 0.05),PBS group(79.6 ± 6.2%,p < 0.05).Fewer immunoreactive microvessels were observed in the tumor tissue sections of Lgr5-DC-CD8~+ CTL treated mice(2.1 ±0.3%)compared to the DC-CD8~+ CTL group(4.2 ± 0.8%,p < 0.05)and the PBS group(8.7 ± 0.7%,p < 0.05);The expression of VEGF protein in the Lgr5-DC-CD8~+ CTL group was 30.7±4.0 %,that was actually significantly lower than that of the DC-CD8~+ CTL group(75.7 ±3.2%,p < 0.01),or PBS group(80.0 ± 5.0%,p < 0.01).HE staining showed that Lgr5-DC-CD8~+ CTL treatment resulted in significant pathological changes in tumor tissues.Conclusion: Lgr5 protein stimulated the maturation of DC cells that induced the production of Lgr5 antigen specific CD8~+ CTL.Lgr5-DC-CD8~+ CTL can effectively kill tumor cells and delay the growth of tumor,which is of great significance for the immunotherapy of colon cancer.The mechanism of Lgr5-DC-CD8~+ CTL inhibiting tumor of colorectal cancer might inhibit cell proliferation through its action on Ki-67,and decreased micro-vessel density(MVD)associated with CD31 and vascular endothelial growth factor(VEGF). |
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