肺鳞癌引流区域淋巴结细胞对自体肿瘤凋亡相关蛋白表达水平的影响

Effect of cells derived from tumor-draining lymph nodes of lung cancer patients on the expressions of apoptosis-related proteins in autologous tumor cells

目的：研究肺癌引流淋巴结（tumor draining lymph nodes，TDLN）细胞对自体肿瘤的凋亡相关蛋白表达水平的影响。方法：手术时取肺癌患者肿瘤引流淋巴结2～4枚，制备细胞悬液，分别用IL-2（I-TDLN）、IL-2＋GM-CSF＋IL-4（G-TDLN）刺激后，对建立人肺癌细胞移植瘤模型的裸鼠进行免疫干预，检测不同组裸鼠移植瘤细胞的凋亡率和Bcl-2、Bax、survivin、Fas、FasL蛋白的表达。结果：对照组荷瘤裸鼠癌组织的凋亡率为（6．07±3．31）％，IL-2处理组的凋亡率为（12．11±1．19）％；LAK细胞组的凋亡率为（21．42±1．82）％。I-TDLN细胞组的凋亡率为（24．60±0．96）％，G-TDLN细胞组的凋亡率为（32．76±4．46）％。方差分析显示各组间肿瘤组织的凋亡差异显著（P〈0．001）。Fas及FasL蛋白在空白对照组及IL-2、LAK、I-TDLN、G-TDLN干预组移植瘤细胞表达的FI值均无统计学差异（P=0．246，P=0．528）。然而经LAK、I-TDLN、G-TDLN干预后。移植瘤细胞Bax蛋白的表达FI值明显高于空白对照组及IL-2作用组（P〈0．001）；而Bcl-2蛋白在IL-2、LAK、I-TDLN和G-TDLN干预组小鼠移植瘤细胞中的表达FI值明显低于空白对照组（P〈0．001）；同样survivin蛋白在LAK、I-TDLN和G-TDLN干预组移植瘤细胞中的表达FI值明显低于空白对照组及IL-2作用组（P〈0．001）。结论：肺癌引流淋巴结细胞对自体肿瘤的杀伤可能与凋亡蛋白有关。

Objectives： To study the effect of cells derived from tumor-draining lymph nodes （TDLN） of lung cancer patients on the expressions of apoptosis-related proteins in autologous tumor cells. Methods： Two to four tumor-draining lymph nodes without metastasis were collected from lung cancer patients during surgery. The TDLN cells were suspended in RPMI-1640 medium and stimulated with IL-2 175 U/mL （ I-TDLN group） or IL-2 175 U/mL ＋ GM-CSF ＋ IL-4 500 U/mL （ D-TDLN group） in culture flask, and then incubated at 37℃ with 5% CO2. The human lung cancer cells were subcutaneously implanted into nude mice to establish tumor xenograft model. Then the nude mice were treated with TDLNs. The apoptotic rate and the expressions of Fas/FasL, Bax, Bcl-2, and survivin protein in xenograft tumors were analyzed by flow cytometry analysis. Results： The apoptotic rate of lung cancer cells was （ 6.07±3.31）%, （12.11±1.19）%, （21.42±1.82）%, （24.60±0.96）%, and （32.76±4.46）% in blank control group, IL-2 group, LAK ceils group, I-TDLN group, and G-TDLN group, respectively. ANOVA analysis showed there was significant difference in apoptotic rates between each group （P 〈0.001 ）. The fluorescence intensity （FI） of Fas protein was 1.46±0.70, 1.47±0.72, 1.44±0.43, 1.42±0.30, and 1.40±0.45 in blank control group, IL-2 group, LAK cells group, I-TDLN group and G-TDLN group, respectively （P =0.246）. The FI value of FasL protein was 1.53±0.29, 1.56±0.41, 1.58±0.56, 1.56±0.36 and 1.57±0.81 in blank control group, IL-2 group, LAK cells group, I-TDLN group, and G-TDLN group, respectively. The difference was not significant （ P =0. 528 ）. The FI value of Bax protein was significantly higher in LAK group, I-TDLN group and G-TDLN than that in blank control and IL-2 group in blank control group （ 1.66±0.84, 1.72±0.53 and 1.87±0.55 vs 1.36±0.39 and 1.37±0. 16, respectively, P 〈0.001 ）. The FI value of Bcl-2 protein was significantly lower in IL-2 group, LAK cells group, I-TDLN group, and G-TDLN group than that of blank control （ 1.39±0.40, 1.22±0.14, 1.15±0.19 and 1.06±0.75 vs 1.70±0.88, respectively, P 〈 0.001 ）. The FI value of survivin protein was significantly lower in LAK group, I-TDLN group and G-TDLN than that in blank control and IL-2 group （1.54±0.62, 1.45±0.61 and 1.35±0.36 vs 1.71±0.71 and 1.69±0.20, respectively, P 〈0.001）. Conclusion： The autologous tumor-specific killing effect of TDLN cells is related with regulation of apoptosis-related proteins.