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目的探讨汉黄芩素对人NK细胞杀伤胃癌MKN45细胞的影响及作用机制。方法淋巴细胞分离液分离外周血单个核细胞(PBMC),在体外经rhIL-2诱导培养NK细胞;不同浓度的汉黄芩素分别作用于NK细胞24 h、48 h及72 h后CCK8法检测NK细胞增殖情况;不同浓度汉黄芩素作用NK细胞48 h后:流式细胞术(FCM)检测NK细胞GraB、IFN-γ、PFP、CD107a的表达;Western blot检测NK细胞p-Akt、β-catenin、Bcl-2、p-ERK的表达;乳酸脱氢酶(LDH)释放法检测NK细胞对胃癌细胞株MKN45的杀伤活性。结果 0.2~50μg/ml的汉黄芩素作用NK细胞24 h、48 h及72 h后,NK细胞的增殖率增高;同一质量浓度汉黄芩素作用48 h后的NK细胞的增殖率最高且在12.5μg/ml时达最高峰;汉黄芩素3.1~12.5μg/ml作用NK细胞48 h后:NK细胞的GraB、IFN-γ、PFP、CD107a表达增加、NK细胞的p-Akt、β-catenin表达增加、NK细胞对胃癌MKN45细胞的杀伤活性增高。结论汉黄芩素能够促进NK细胞的增殖,其促进NK细胞的增殖可能与Wnt信号传导通路及PI3K/Akt信号通路有关;汉黄芩素增加NK细胞对胃癌细胞的杀伤活性可能与汉黄芩素上调NK细胞GraB、PFP、IFN-γ及CD107a的表达有关。
Objective To investigate the effect and the mechanism of wogonin on human NK cells killing gastric cancer MKN45 cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by lymphocyte separation and induced by rhIL-2 in vitro. Different concentrations of wogonin were applied to NK cells for 24 h, 48 h and 72 h, respectively. After NK cells were treated with different concentrations of wogonin for 48 h, the expression of GraB, IFN-γ, PFP and CD107a in NK cells were detected by flow cytometry (FCM). The expressions of p-Akt and β-catenin , Bcl-2, p-ERK expression; NK cell killing activity of gastric cancer cell line MKN45 was detected by lactate dehydrogenase (LDH) release assay. Results The proliferation of NK cells was increased after treated with wogonin at 0.2-50 μg / ml for 24 h, 48 h and 72 h. The highest proliferation rate of NK cells was observed at 48 h after the same concentration of wogonin μg / ml, respectively. After treated with WBC at 3.1 ~ 12.5μg / ml for 48 h, the expression of GraB, IFN-γ, PFP and CD107a in NK cells increased and the expressions of p-Akt and β-catenin Increased, NK cells on gastric cancer MKN45 cells increased cytotoxic activity. Conclusion Wogonin can promote the proliferation of NK cells and promote the proliferation of NK cells, which may be related to the Wnt signaling pathway and the PI3K / Akt signaling pathway. Wogonin could increase the cytotoxic activity of NK cells to gastric cancer cells, Cell GraB, PFP, IFN-γ and CD107a expression.