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Archive > Volume 27 Issue 3 > 2020,27(3):221-227. DOI:10.3872/j.issn.1007-385X.2020.03.002 Prev Next
Basic Research
Anti-tumor effect of CTL on colon cancer xenograft in nude mice after blockingout CTLA-4 with CRSIPR/Cas9 technology
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Abstract:
Objective: To investigate the anti-tumor effect of CTL cells on colon cancer xenograft in nude mice after knocking out the immune check point CTLA-4 by CRISPR/Cas9 technology. Methods: A specific small guide RNA (sgRNA) for CTLA-4 was designed to construct sgRNA/Cas9 plasmid, which was then transfected into CTL using a lentiviral vector to obtain CTL cells with CTLA-4 deletion (CTLA-4 KO CTL). The transfection efficiency of the plasmid and the deletion efficiency of CTLA-4 were verified. BALB/c nude mice were randomly divided into two groups to prophylactically inoculate CTLA-4 KO CTL (experimental group) or CTL (control group); 3 days later, the animals of two groups were inoculated with colon cancer cell line LS174-T to observe the tumor formation rate and tumor formation time. After constructing colon cancer xenograft model in nude mice, the animals were randomly divided into two groups, respectively treated with CTLA-4 KO CTL (experimental group) and CTL (control group) cells to observe the tumor growth volume and survival time of mice. The serum levels of TNF-α and IFN-γ in nude mice were detected. Results: sgRNA was designed and CRSIPR/Cas9 system with lentivirus as vector was successfully constructed. CTL cells were transfected with the established CRSIPR/Cas9 system, and the highest transfection efficiency was up to (28.80±0.62)%. After transfection, the deletion efficiency of CTLA-4 was detected by Flow cytometry. The CTLA-4 expression of CTLA-4 KO CTL group was significantly lower than that of CTL group [(0.91±0.25)% vs (42.70±2.72)%, P<0.05]. In prophylactic assay, the formation rate of colon cancer xenografts in the experimental group was significantly lower than that in the control group (33.33%vs 100%, P<0.05). In treatment assay, the tumor volume in the experimental group was significantly inhibited compared with the control group ([503±23.9] vs [911.2±51.4] mm3, P<0.05), and the survival time of the experimental group was significantly prolonged (median survival time: 78 d vs 42 d, P<0.05); Moreover, the secretion levels of serumTNF-α ([268.93±17.04] pg/ml vs [148.26±20.07] pg/ml, P<0.05) and IFN-γ (315.38±18.67 pg/ml vs 202.92±29.32 pg/ml,P<0.05) in the experimental group were significantly higher than those in the control group. Conclusions: The lentiviral vector CRSIPR/Cas9 system is an effective gene editing method; its successful deletion of CTLA-4 in CTL cells can significantly inhibit the tumor formation rate of colon cancer xenografts in nude mice and enhance the anti-tumor effect of CTL on colon cancer xenografts.
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Project Supported:
Project supported by the Key Medical Science Research of Hebei Province (No.20180375)
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