p53凋亡刺激蛋白在非小细胞肺癌中的表达及其临床意义

The expression and clinical significance of apoptosis stimulating protein of p53 in nonsmall-cell lung cancer

目的研究分析p53遗传背景不同的非小细胞肺癌（NSCLC）细胞株中p53凋亡刺激蛋白（ASPP）家族的表达和调节，以及在NSCLC诊断和化疗监测中的临床意义。方法用实时荧光定量逆转录（FQ—RT）-PCR检测NSCLC细胞株A549（野生型）和NCI—H157（突变型）及37例肺癌患者组织与外周血单个核细胞中ASPP1、ASPP2mRNA表达，免疫印迹技术检测NSCLC细胞株ASPP1、ASPP2蛋白含量。同时，检测2株细胞对顺铂（CDDP）的敏感性以及化疗药物处理后ASPP1、ASPP2mRNA表达变化。此外，还比较NSCLC患者与37名健康对照者ASPP1、ASPP2mRNA表达情况，并监测NSCLC患者化疗前后ASPP1、ASPP2mRNA表达变化。结果FQ—RT-PCR检测ASPP标准品曲线斜率均值分别为-3．249、-3．358，r〉0．98，标准曲线的扩增效率分别为1．156、1．028；CDDP处理后NCI-H157细胞株的Ic50值是3．70μg/ml，A549的Ic50是10．48μg/ml。NCI—H157细胞株中ASPP1，ASPP2mRNA平均表达量比A549分别高2．66倍和6．98倍。肺癌患者组织和外周血单个核细胞中ASPP1mRNA表达分别为（4．27±0．57）×10^3和（2．49±0．32）×10^3，ASPP2 mRNA表达分别为（2．34±0．75）×10^3和（7．00±1．17）×10^3，而健康对照组组织和外周血单个核细胞中ASPP1 mRNA表达分别为（1．32±0．21）×10^4和（1．46±0．31）×10^4，ASPP2 mRNA表达分别为（1．38±0．19）×10^4和（1．28±0．18）×10^4。肺癌组中ASPP1和ASPP2mRNA明显低于健康对照组（t值分别为2．58、3．94、3．62、3．76，P均〈0．05）。NSCLC组中18例患者术后化疗前ASPP1和ASPP2mRNA分别为（2．34±0．56）×10^3和（6．64±0．72）×10^3，进行2个周期化疗后分别为（3．66±0．64）×10^3和（9．42±4-0．44）×10^3，基因表达量均明显升高（t值分别为3．02、4．50，P均〈0．05）。结论NSCLC突变型细胞株ASPPI和ASPP2 mRNA的表达均高于野生型细胞株，ASPP1和ASPP2表达升高有可能增强肿瘤细胞对化疗药物的敏感性。NSCLC患者化疗过程中，ASPP1和ASPP2mRNA表达的变化可能与化疗药物作用有关，提高ASPP1、ASPP2的表达将有助于肿瘤的治疗。

Objective To investigate the expression and regulation of ASPP family mRNA in NSCLC cell lines, and study the diagnostic and therapeutic significances of apoptosis stimulating of p53 expression in NSCLC patients. Methods Real-time fluorescence quantitative PCR was established and used to measure the expression levels of ASPP mRNA in NSCLC cell lines A549 （wild-type p53） and NCl-H157 （mutant-type p53） with p53 different genetic background, and ASPP mRNA was also detected in NSCLC tissue, peripheral blood of 37 NSCLC patients. Western Bloting was used to examine the levels of ASPP1 and ASPP2 proteins of NSCLC cell lines. Chemosensitivity of the cell lines to CDDP was analyzed by MTT assay, and detect the change of ASPP1 and ASPP2. Compared with the ASPPs mRNA expression between NSCLC patients and healthy controls, and monitoring ASPPs mRNA expression in patients with NSCLC chemotherapy. Results The slope rates of standard curves were -3. 249, -3. 358 respectively, and correlation coefficients were more than 0. 98. Amplification efficiency of standard curves were 1. 156,1. 028 respectively. The IC50 values of NCI-H157 and A549 cells were 3.70, 10. 48 μg/ml respectively. Mean ASPP1 ASPP2 mRNA levels were a statistically significant 2. 66 folds and 6. 98 folds higher in NCI-H157 cell compared to A549 cell. The levels of ASPP1 mRNA in tumor tissues and peripheral blood were （4.27±0. 57）×10^3, （2.49±0. 32）×10^3 in patients with NSCLC respectively, and the levels of ASPP2 mRNA in tumor tissues and peripheral blood were （2. 34±0. 75 ）×10^3, （7.00±1.17）×10^3 in patients with NSCLC respectively. The levels of ASPP1 and ASPP2 mRNA in tissues and peripheral blood were （ 1.32±0. 21 ）×10^4, （ 1.46±0. 31 ）× 10^4 and （ 1.38v0. 19）×10^4, （ 1.28±0. 18）×10^4 in control groups respectively. The ASPP1, ASPP2 mRNA levels in patients with NSCLC were significantly lower than those of control groups （t =2. 58, 3.94, 3.62, 3.76, P 〈0. 05）. Before chemotherapy the levels of ASPP1 and ASPP2 mRNA in patients with NSCLC were （2. 34±0. 56）×10^3 and （6. 64±0.72）×10^3 ,and after 2 periods of postoperative chemotherapy the levels were （3.66±0. 64）×10^3 and （9.42±0. 44） ×10^3, the expression was higher than those of chemotherapy before （t = 3.02, 4. 50, P 〈 0. 05）. Conclusions The expression of ASPP1, ASPP2 mRNA in p53 mutant-type cell lines is higher than that in p53 wild-type cell lines. The expression of ASPP maybe has close relationship with chemotherapeutic sensitivity of NSCLC. In NSCLC patient chemotherapy process, the change of ASPP1 and ASPP2 mRNA expression may be related with chemotherapy medicine function. Enhance the expression of ASPP1 and ASPP2 will be beneficial to the tumor treatment.