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目的探讨糖原合酶激酶3β抑制剂4,6-二取代吡咯并嘧啶(TWS119)调控Wnt/β-catenin信号通路对NK细胞增殖和表型的影响。方法分离健康人外周血单个核细胞(PBMC),加入到含重组人IL-2和人AB血清的NK细胞完全培养基中诱导培养,获得NK细胞。(0~8.0)μmol/L TWS119处理NK细胞72 h,Western blot法检测NK细胞β-catenin的表达、CCK-8法测定NK细胞增殖能力;流式细胞术检测各组NK细胞CD107a和CD62L(L-选择素)的表达。结果培养10 d后的NK细胞纯度达到(61.76±3.74)%;TWS119能够活化NK细胞Wnt/β-catenin信号通路。(0~2.0)μmol/L TWS119处理NK细胞,能显著促进NK细胞增殖,浓度大于2.0μmol/L时增殖率逐渐降低。(0~8.0)μmol/L TWS119处理NK细胞,CD62L的阳性表达率逐渐升高,且呈剂量依赖性;与之相反,CD107a阳性表达率逐渐下降。结论 TWS119活化NK细胞Wnt/β-catenin信号通路可获得早期成熟的CD62L+NK细胞。
Objective To investigate the effect of Wnt / β-catenin signaling pathway on the proliferation and phenotype of NK cells regulated by glycogen synthase kinase 3β inhibitor 4,6-disubstituted pyrrolopyrimidine (TWS119). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood mononuclear cells (PBMCs) and cultured in complete medium of NK cells containing recombinant human IL-2 and human AB serum to obtain NK cells. (0 ~ 8.0) μmol / L TWS119 for 72 h. The expression of β-catenin in NK cells was detected by Western blot. The proliferation of NK cells was assayed by CCK-8 assay. Flow cytometry was used to detect the expression of CD107 a and CD62L L-selectin) expression. Results The purity of NK cells reached (61.76 ± 3.74)% after cultured for 10 days. TWS119 could activate the Wnt / β-catenin signaling pathway in NK cells. (0 ~ 2.0) μmol / L TWS119 could significantly promote the proliferation of NK cells. When the concentration was more than 2.0μmol / L, the proliferation rate of NK cells decreased gradually. (0 ~ 8.0) μmol / L TWS119 treatment of NK cells, CD62L positive expression rate gradually increased, and in a dose-dependent manner; In contrast, CD107a positive expression rate decreased gradually. Conclusion TWS119 activates NK cell Wnt / β-catenin signaling pathway to obtain early matured CD62L + NK cells.