石见穿多糖增强γδT细胞对骨肉瘤细胞的杀伤作用

Polysaccharides from Salvia chinensis Benth enhance the killing effect of γδT cells against osteosarcoma cells

目的探讨石见穿多糖(polysaccharides from salvia chinensis,PSSC)作用对γδT细胞增殖以及对骨肉瘤细胞杀伤活性的影响及其分子机制。方法分别采用不同质量浓度的PSSC处理γδT细胞和骨肉瘤细胞24、48、72 d,然后以细胞计数试剂盒(CCK-8)检测PSSC对γδT细胞和骨肉瘤细胞增殖的影响,确定PSSC最佳诱导条件。后续实验均采用最佳条件诱导γδT细胞。酶连免疫吸测定(ELISA)检测PSSC诱导前后γδT细胞肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)的水平;流式细胞术细胞术检测γδT细胞穿孔素、颗粒酶素B、CD107a和NK细胞受体G2D(NKG2D)的表达变化。乳酸脱氢酶释放法检测PSSC对γδT细胞杀伤骨肉瘤细胞活性的影响。流式细胞术检测PSSC对γδT细胞诱导骨肉瘤细胞凋亡作用的影响。结果 5.00、10.00、20.00、40.00 mg/L的PSSC均可促进γδT细胞增殖,以20.00 mg/L的PSSC诱导48 h时作用最为明显(P<0.05)。经PSSC诱导后的γδT细胞TNF-α、IFN-γ、穿孔素、颗粒酶素B、CD107a和NKG2D的表达显著高于对照组(P<0.05),对骨肉瘤细胞的杀伤率和诱导凋亡率显著高于对照组(P<0.05)。结论石见穿多糖能够促进γδT细胞增殖,并增强其对骨肉瘤细胞的杀伤作用和凋亡诱导作用,其机制可能与促进细胞表面穿孔素、颗粒酶素B、CD107a和NKG2D的表达和增加TNF-α和IFN-γ的分泌有关。

This study was performed to explore the effect of polysaccharides from Salvia chinensis Benth(PSSC) on the proliferation of γδT cells and the killing activity of γδT cells on osteosarcoma cells and the molecular mechanism. γδT cells and osteosarcoma cells were cultured respectively with different concentrations of PSSC for24, 48, and 72 h. The effect of PSSC on the proliferation of γδT cells and osteosarcoma cells were determined by CCK-8 method, so as to determine the optimal conditions for PSSC induction. ELISA was used to detect the levels of TNF-α and IFN-γ before and after the induction of PSSC. The expression changes of perforin, granzyme B, CD107 a and NKG2 D were detected by flow cytometry. Lactate dehydrogenase release method was used to detect the effect of PSSC on the activity of γδT cells in killing osteosarcoma cells. Flow cytometry was used to detect the effect of PSSC on the induction of osteosarcoma cell apoptosis by γδT cells. Data showed that PSSC(5.00, 10.00, 20.00, and 40.00 mg/L) could promote the proliferation of γδT cells, especially at 20.00 mg/L for 48 h(P<0.05). The expressions of TNF-α, IFN-γ, perforin, granzyme B, CD107 a, and NKG2 D in γδT cells induced by PSSC were significantly higher than those in the control group(P<0.05);PSSC(20.00 mg/L for 48 h) also promoted the killing activity and apoptosis inducing ability of γδT cells against osteosarcoma cells, as compared with the control group(P<0.05). In conclusion,PSSC could promote the proliferation of γδT cells and enhance their killing and apoptosis-inducing effects on osteosarcoma cells. The mechanism may be related to promoting the expression of perforin, granzyme B,CD107 a and NKG2 D on the cell surface and increasing the secretion of TNF-α and IFN-γ.