金丝桃苷对NK细胞杀伤人胰腺癌PANC1细胞的增强效应

Enhancement effects of hyperoside on killing activity of human NK cells against pancreatic cancer PANC1 cells

目的观察金丝桃苷对人NK细胞体外增殖及杀伤胰腺癌PANC1细胞活性的影响，探讨其可能机制。方法分离健康人外周血单个核细胞（PBMCs），应用含IL-2的NK细胞培养液加不同浓度（0.3、1.6、8、40、200 μg/ml）的金丝桃苷溶液诱导PBMCs 12 d，以不加金丝桃苷作为对照组。采用台盼蓝染色观察细胞存活数；流式细胞术检测NK细胞表型及其穿孔素、颗粒酶B的表达，计算NK细胞增殖倍数；乳酸脱氢酶（LDH）释放法检测NK细胞杀伤活性。结果金丝桃苷各浓度组和对照组NK细胞含量均达到80%左右；NK细胞表型为CD3－CD56＋，0.3、1.6、8 μg/ml金丝桃苷组NK细胞增殖倍数分别为（93.76±8.77）、（106.67±12.35）、（118.50±11.51）倍，显著高于对照组的（73.70±9.43）倍；1.6、8、40 μg/ml金丝桃苷组NK细胞穿孔素水平显著高于对照组[ （82.34±2.90）%、（89.15±3.54）%、（81.78±2.81）%比（72.93±2.06）% ]；1.6、8 μg/ml金丝桃苷组NK细胞颗粒酶B水平显著高于对照组[（87.30±1.70）%、（92.16±3.05）%比（82.35±2.73）% ]；1.6、8 μg/ml金丝桃苷组对PANC1细胞的杀伤活性显著高于对照组[（63.18±3.77）%、（65.34±4.97）%比（52.16±5.48）%]，差异均有统计学意义（P值均〈0.05）。结论金丝桃苷在一定浓度范围内可促进NK细胞增殖，并可能通过上调穿孔素和颗粒酶B的表达增强NK细胞对胰腺癌PANC1细胞的杀伤活性。

Objective To investigate the effect of hyperoside on proliferation and killing activity of NK cells against pancreatic cancer PANC1 cells in vitro, and explore its potential mechanism.Methods Peripheral blood mononuclear cells of healthy donors were isolated, NK cells were induced with medium contained with IL-2 and different concentrations of hyperoside （0.3, 1.6, 8, 40 and 200 μg/ml） for 12 days. Cell viability was observed by trypan blue staining. Phenotype and perforin, granzyme B expression of NK cells were detected by flow cytometry. Killing activity of NK cells against PANC1 cells were analyzed with lactate dehydrogenase （LDH） releasing method.Results The proportion of NK cells in control group and experimental group treated with different concentration of hyperoside both reached about 80%, respectively. The proliferation of CD3-CD56＋ NK cells treated by hyperoside at 0.3, 1.6 and 8 μg/ml was （93.76±8.77）, （106.67±12.35） and （118.50±11.51） times, respectively, which were significantly higher than （73.70±9.43） times of the control group. The expressions of perforin in NK cells treated with hyperoside at 1.6, 8 and 40 μg/ml were significantly higher than those of the control group [（82.34±2.90）%, （89.15±3.54）%, （81.78±2.81）% vs （72.93±2.06）%]. The expressions of granzyme B in NK cells treated with hyperoside at 1.6 and 8 μg/ml were significantly higher than those of the control group [（87.30±1.70）%, （92.16±3.05）% vs （82.35±2.73）%]. The killing activity of NK cells against PANC1 cells treated by hyperoside at 1.6 and 8 μg/ml was significantly higher than those of the control group [（63.18±3.77）%, （65.34±4.97）% vs （52.16±5.48）%]. The differences were statistically significant （all P〈0.05）.Conclusions Hyperoside could promote the proliferation of NK cells at certain concentrations and maybe enhance the killing effect against pancreatic cancer PANC1 cells through up-regulating the expression of perforin and granzyme B in NK cells.