低浓度紫杉醇对NK-92MI细胞增殖及细胞毒效应的影响  

Effect of low concentration paclitaxel on cytotoxicity of NK-92MI cells

作　　者：曹飞麟[1] 郑瑞[2] 周申康[1] 马兆生[1] 林辉[1] 徐东[1] 

机构地区：[1]浙江省台州医院肿瘤外科,317000 [2]浙江省台州医院中心实验室,317000

出　　处：《浙江医学》2012年第6期431-434,437,共5页Zhejiang Medical Journal

基　　金：台州市科技计划项目（A102KY09）;浙江省医药卫生科技计划项目（2011KYB137）

摘　　要：目的 探讨低浓度肿瘤化疗药物紫杉醇对人恶性非霍奇金淋巴瘤患者的自然杀伤细胞（NK-92MI）细胞系生长及细胞毒效应的影响.方法 体外培养NK-92MI,加入不同浓度（1、0.5、0.25、0.125、0.06、0.03、0.015、0.008、0.004μmol/L）的紫杉醇培养72h,MTS法检测NK-92MI细胞的增殖;以低浓度（0.02μmol/L）的紫杉醇预培养NK-92MI细胞48h,杀伤试剂盒检测NK-92MI 细胞对K-562细胞的细胞毒效应,流式细胞术检测NK-92MI细胞表面NKG2D受体的表达,RT-PCR检测NK-92MI受体NKG2D、NKG2A/B以及效应分子颗粒酶、穿孔素及IFN-γ基因的表达,ELISA检测IFN-γ分泌的改变.结果 高浓度（0.06～1μmol/L）紫杉醇明显抑制细胞的生长（P＜0.05）,且呈浓度梯度依赖性;低浓度（0.008～0.06μmol/L）紫杉醇能相对促进NK-92MI细胞的增殖,但无统计学意义（P＞0.05）,当低于0.008μmol/L时,对NK-92MI细胞的增殖不影响（P＞0.05）.0.02μmol/L紫杉醇能增强NK-92MI细胞的细胞毒活性,在效靶比为10∶1、5∶1、2.5∶1、1.25∶1时,实验组细胞毒效应均明显高于对照组（P＜0.05或0.01）.紫杉醇处理组NK-92MI细胞高表达穿孔素、颗粒酶及IFN-γ（均P＜0.05）,而抑制性受体NKG2A/B基因表达下调（P＜0.05）,未发现表面杀伤性受体NKG2D基因和蛋白的表达上调（P ＞0.05）.结论 低浓度紫杉醇能促进NK-92MI细胞的增殖和细胞毒活性,细胞毒活性的增强可能与紫杉醇处理后NK-92MI细胞活化能力增强以及相关基因（穿孔素和颗粒酶）的表达增强有关.Objective To investigate the effect of low concentration paclitaxel on cytotoxicity of NK-92MI cells. Methods The cultured NK-92MI cells were treated with different concentrations of paclitaexl （0.004 -1.0μmol/L）for 72h and the cell proliferation was test by MTS method. NK-92MI cells were pretreated with low concentration paclitaxel （0.02 μ mol/L）for 48h; the cytotoxic effect was analyzed with cytotoxicity assay kit and the expression of NKG2D receptor at NK-92MI cell surface was assessed by flow cytometry. The expressions of NKG2D, NKG2A/B, perforin, granzyme-B and IFN- γ mRNAs were determined by RT-PCR. the IFN-γ level in culture supernatant was measured by ELISA method. Results The proliferation of NK-92MI cells treated with higher concentration paclitaxel （0.06 - 1.0 IJ mol/L） for 72h was inhibited significantly（P〈0.05 ）, but not affected by low concentration paclitaxel （0.008 - 0.06 p mol/L）. Pretreatment of 0.02 p mol/L paclitaxel for 48h enhanced cytotoxicity of NK-92MI cells against K562 cells （P〈0.05）.The mRNAs of perforin, granzyme-B and IFN-y were up-regulated in a time dependent manner, while the expression of NK cell inhibitory receptor NKG2A/B was down-regulated, and there was no difference both in NKG2D mRNA and membrane protein expression between paclitaxel treated group and control. Conclusion Low concentration paclitaxel enhances cytotoxicity of NK92-MI cells against target K-562 cells, which may be associated with disturbing the balance between inhibitory and activating receptors followed by increased perforin and granzyme-B production.

关 键 词：紫杉醇 NK-92MI细胞 干扰素-γ NKG2D受体 NKG2A/B受体 颗粒酶 穿孔素 

分 类 号：R392.11[医药卫生—免疫学]