【摘要】 目的探讨舒尼替尼诱导自然杀伤细胞2族成员D(NKG2D)配体(NKG2DL)的表达对自然杀伤(NK)细胞杀伤舌鳞癌CAL27细胞敏感性的影响。方法将对数生长期的CAL27细胞分为未处理组和舒尼替尼组,舒尼替尼组采用舒尼替尼处理,未处理组细胞未经舒尼替尼处理。采用细胞克隆形成实验检测两组CAL27细胞的增殖能力;采用流式细胞术检测两组CAL27细胞的凋亡、NKG2DL(MICA、MICB、ULBP1、ULBP2和ULBP3)阳性细胞的表达及健康人NK细胞的纯度;采用免疫蛋白印迹法检测两组CAL27细胞中NKG2DL(MICA、MICB、ULBP1、ULBP2和ULBP3)蛋白的表达水平。再将舒尼替尼组分为舒尼替尼-NKG2D组和舒尼替尼-对照组,舒尼替尼-NKG2D组加入经NKG2D单抗处理后的NK细胞,舒尼替尼-对照组加入未经NKG2D单抗处理的NK细胞,同时未处理组加入未经NKG2D单抗处理的NK细胞(作为未处理-NK组)。采用乳酸脱氢酶释放法检测NK细胞对CAL27细胞的杀伤敏感性。结果 (1)与未处理组相比,舒尼替尼组CAL27细胞的增殖能力降低、凋亡率增加,CAL27细胞中的MICA、MICB、ULBP1和ULBP2蛋白表达水平均升高(均P<0.05),而两组CAL27细胞ULBP3蛋白表达水平差异无统计学意义(P>0.05)。(2)与未处理-NK组相比,NK细胞对经舒尼替尼处理的CAL27细胞的杀伤敏感性增强(均P<0.05)。(3)采用NKG2D抗体封闭NK细胞表面的NKG2D受体后,NK细胞对经舒尼替尼处理的CAL27细胞的杀伤敏感性低于未采用NKG2D单抗处理的CAL27细胞的杀伤敏感性(均P<0.05)。结论舒尼替尼可以抑制舌鳞癌细胞CAL27细胞的增殖,促进其凋亡,上调CAL27细胞中NKG2DL(MICA、MICB、ULBP1、ULBP2)的表达水平,增强NK细胞对CAL27细胞的杀伤敏感性。更多还原

【Abstract】 Objective To investigate the effect of sunitinib-induced natural killer group 2 member D ligand(NKG2 DL) expression on the killing sensitivity of natural killer(NK) cells to tongue squamous cell carcinoma CAL27 cells.Methods CAL27 cells in logarithmic phase were divided into non-treatment group and sunitinib group. The sunitinib group received sunitinib for treatment, while the non-treatment group did not receive sunitinib for treatment. In both groups, cell colony formation assay was used to detect the proliferation ability of CAL27 cells; flow cytometry was employed to detect the apoptosis of CAL27 cells, the expression of NKG2 DL(MICA, MICB, ULBP1, ULBP2 and ULBP3)-positive cells, and the purity of healthy people′s NK cells; Western blotting assay was used to detect the expression level of NKG2 DL(MICA, MICB, ULBP1, ULBP2 and ULBP3) protein in CAL27 cells. Then the sunitinib group was further divided into sunitinib-NKG2 D group and sunitinib-control group. The NK cells treated with NKG2 D monoclonal antibody were added into the sunitinib-NKG2 D group, while the NK cells not treated with NKG2 D monoclonal antibody were added into the sunitinib-control group. At the same time, the NK cells not treated with NKG2 D monoclonal antibody were added into the non-treatment group(non-treatment-NK group). Lactate dehydrogenase releasing assay was adopted to determine the killing sensitivity of NK cells to CAL27 cells.Results(1)Compared with the non-treatment group, the sunitinib group exhibited a decreased proliferation ability and an increased apoptosis rate of CAL27 cells, together with elevated expression levels of MICA, MICB, ULBP1, and ULBP2 proteins in CAL27 cells(all P<0.05), whereas there was no statistically significant difference in the expression level of ULBP3 protein in CAL27 cells between the two groups(P>0.05).(2) Compared with the non-treatment-NK group, NK cells had an increased killing sensitivity to sunitinib-treated CAL27 cells(all P<0.05).(3) NK cells harbored a lower killing sensitivity to sunitinib-treated CAL27 cells than to CAL27 cells without NKG2 D monoclonal antibody treatment after NKG2 D receptors on NK cells surface had been blocked by NKG2 D antibody(all P<0.05).Conclusion Sunitinib can inhibit the proliferation of tongue squamous cell carcinoma CAL27 cells, promote their apoptosis, up-regulate the expression level of NKG2 DL(MICA, MICB, ULBP1, ULBP2) in CAL27 cells, and enhance the killing sensitivity of NK cells to CAL27 cells.更多还原