高频热疗对人肿瘤细胞NKG2D配体表达影响的研究

Effect of high frequency hyperthermia on the expression of NKG2D ligandin human tumor cell line

目的:探讨高频热疗(HFH)对人乳腺癌MDA-MB-231细胞和肺癌XWLC-05细胞NKG2D配体表达的影响。方法:用工作频率为13.56MHz HFH仪处理人乳腺癌MDA-MB-231细胞和肺癌XWLC-05细胞,参数为温度43℃,最大输出功率1 200W,维持输出功率60%～80%。每次处理时间为40min,1次/d。分别提取未处理、HFH处理1次和3次的肿瘤细胞mRNA,逆转录成cDNA,用荧光定量PCR检测NKG2D配体mRNA的表达,并用流式细胞术检测肿瘤细胞表面NKG2D配体蛋白质表达。结果:荧光定量PCR结果显示,MDA-MB-231细胞表达MICA、MICB、ULBP2和ULBP3mRNA,HFH处理1次后,MICA、MICB、ULBP2和ULBP3表达分别上调1.44、1.43、1.88和2.06倍,其中MICB表达上调差异有统计学意义,P<0.05;处理3次后,分别上调1.78、2.00、2.13和2.91倍,差异均有统计学意义,P<0.05。在HFH处理前后,MDA-MB-231细胞都不表达ULBP1;HFH处理3次后,XWLC-05细胞MICA、MICB、ULBP2和ULBP3mRNA的表达增加,其中,ULBP1和ULBP3的mRNA表达显著上调18.54和2.67倍,P<0.05。HFH处理后,流式细胞分析结果表明,MDA-MB-231和XWLC-05细胞表面NKG2D配体蛋白含量与对照组相比差异无统计学意义。结论:人MDA-MB-231和XWLC-05细胞表达NKG2D配体mRNA,HFH上调其NKG2D配体mRNA表达,并且细胞株间有差异性。HFH对这两种细胞表面的NKG2D配体蛋白质影响不明显,其机制需要进一步研究。

OBJECTIVE：To investigate the effect of high frequency hyperthermia （HFH） on NKG2D ligands expres- sion in human breast cancer MDA-MB-231 and lung cancer XWLC-05 cells. METHODS： Both MDA-MB-231 and XWLC-05 cells underwent exposure in high frequency （13.56 MHz） hyperthermia produced by HG-2000 heat therapy in- strument, the instrument was on the order of power：60 %- 80% of 1 200 W, controlled temperature：43 ℃. Both cell lines underwent 40 minutes exposure in HFH once a day. Total RNA was extracted by Trizol protocol from the control and treated cells,and the cDNA was synthesized by reverse transcriptase, mRNA of NKG2D ligands were measured by the re- al-time fluorescence quantitative PCR assay. To further determine whether HFH increased NKG2D ligand protein expression in the cancer cell lines, the ceils after HFH treatments were analyzed for NKG2D ligands on cell surface by PE-conjugated mono- clonal antibodies and flow cytometry. RESULTS： The effect of HFH on NKG2DL mRNA expression in MDA-MB-231 andXWLC-05 cells was assessed by real-time quantitative RT-PCR,the results indicated that MDA-MB-231 cells constitutive- ly expressed mRNA for MICA, MICB, ULBP2 and ULBP3, and up-regulated the gene expression by 1.44,1.43,1.88 and 2.06 folds after once HFH treatment,and by 1.78,2.00,2.13 and 2.91 folds after 3 times treatment （P〈0.05）. We did not find ULBP1 mRNA expression in MDA-MB-231 cells with or without HFH treatment. However,XWLC-05 cells con- stitutively expressed mRNA for MICA, MICB, ULBP1, ULBP2 and ULBP3, significantly up-regulated the ULBP1 and ULBP3 mRNA by 18.54 and 2.67 folds after 3 times HFH treatment（P〈0.05）. NKG2DL protein expression on the cell surface did not show significant difference between control and HFH treatment cells. CONCLUSION.. Both cell lines con- stitutively expressed some NKG2DL mRNAs and up-regulated the mRNA expression significantly after HFH treatment with cell line-specific difference,however,they did not show significant difference in NKG2DL protein on the cell surface on the experimental condition.