人参皂苷Rb1拮抗达沙替尼抑制NK细胞杀伤卵巢癌的研究

Ginsenoside Rb1 Antagonist Dasatinib-induced Inhibition on NK Cells Cytotoxicity to Ovarian Cancer Cells

关键词:  达沙替尼  人参皂苷Rb1  自然杀伤细胞  卵巢癌细胞  杀伤功能

Key words:  dasatinib  ginsenoside Rb1  natural killer cell  ovarian cancer cell  cytotoxicity

摘要:

目的 研究抗癌药达沙替尼对自然杀伤细胞(NK细胞)杀伤卵巢癌细胞的抑制效应，并探讨人参皂苷Rb1拮抗这种免疫抑制效应的作用及其分子机制。方法 分别用达沙替尼、人参皂苷Rb1以及达沙替尼联合人参皂苷Rb1预处理NK细胞，未经药物处理的NK细胞设为对照。采用CFSE/PI双染色法，经流式细胞仪检测与NK细胞混合培养的卵巢癌HO-8910细胞的死亡率来明确NK细胞的杀伤功能；采用Annexin V-FITC/PI双染色法测定NK细胞的凋亡率来明确其生存活力；采用real-time PCR法测定NK细胞颗粒酶B的mRNA表达量；采用免疫印迹法检测NK细胞ERK的蛋白水平。结果 与对照组相比，达沙替尼处理组的NK细胞对卵巢癌HO-8910细胞的杀伤率显著下降(P<0.01)，NK细胞的颗粒酶B的mRNA水平和磷酸化ERK(pERK)表达均下调；达沙替尼合并人参皂苷Rb1处理组的NK细胞对卵巢癌HO-8910细胞的杀伤率较达沙替尼组升高(P<0.05)，而且颗粒酶B的mRNA水平、pERK表达均较达沙替尼组有所恢复；与对照组相比，人参皂苷Rb1单独处理组的NK细胞的生存活力、颗粒酶B水平、pERK表达量以及杀伤功能均无显著差异(P<0.05)。结论 达沙替尼对NK细胞杀伤卵巢癌细胞有抑制效应，可能与下调NK细胞的杀伤介质颗粒酶B和抑制ERK磷酸化有关。人参皂苷Rb1虽然不能直接活化NK细胞，但是能拮抗达沙替尼对NK细胞的上述抑制效应，提示联用人参皂苷Rb1能减少抗癌药物达沙替尼对免疫效应细胞的抑制作用。

Abstract:

OBJECTIVE To analyze the inhibiting effects of dasatinib on natural killer(NK) cells cytotoxicity on ovarian cancer cell and explore the antagonistic effects of ginsenoside Rb1 on dasatinib-induced immunosuppression effects. METHODS The NK cells were pre-treated with dasatinib, ginsenoside Rb1 and dasatinib combined with ginsenoside Rb1, respectively. The NK cells with no drug pretreatment were carried out as control. CFSE/PI double staining was used to measure the death rate of ovarian cancer cell HO-8910 cells after co-culturing with NK cells; Annexin V-FITC/PI staining was used to detect the vitality of NK cells; real-time PCR was applied to evaluate the mRNA level of granzyme B and Western-blotting was used to detect the phosphorylation levels of ERK. RESULTS Compared with the non-pretreated NK cells, the transcription levels of granzyme B, phosphorylation levels of ERK and the cytotoxicity were significantly down-regulated in dasatinib-treated NK cells (P<0.01). Compared with dasatinib treated NK cells, pre-treated in the combination of dasatinib and ginsenoside Rb1, the transcriptional levels of granzyme B, pERK and cytotoxicity of NK cells were elevated(P<0.05). However, the levels of transcriptional granzyme B, pERK and cytotoxicity of NK cells between ginsenoside Rb1 treated group and control group showed no significant difference(P<0.05). CONCLUSION Dasatinib has inhibiting effects on the cytotoxicity of NK cells by decreasing the amount of granzyme B, one of the crucial cytotoxic molecules in NK cells and inhibiting phosphorylation of ERK which is crucial for NK cells reactivity. Though ginsenoside Rb1 can’t activate NK cells directly, it has antagonistic effects on dasatinib-induced immunosuppression of NK cells. It suggests that the combination use of ginsenoside Rb1 and dasatinib can decrease the suppression effects of dasatinib on NK cells.