Zou F, Zhang Z H, Zhang Y T, Zhao J Q, Zhang X L, Wen C L, Song X Y, Zhou W M
Graduate Department of Hebei North University, Zhangjiakou 075000, China.
Department of Respiratory Diseases, the First Attached Hospital of Hebei North University, Zhangjiakou 075000, China.
Zhonghua Zhong Liu Za Zhi. 2017 May 23;39(5):339-343. doi: 10.3760/cma.j.issn.0253-3766.2017.05.004.
To investigate whether cancer-associated- fibroblasts (CAF), the key component of tumor microenvironment, regulate the chemoresistant capacity of lung cancer cell line A549 through SDF-1 secretion. Primary cell isolation techniques was used to isolate cancer-associated-fibroblasts from lung cancer patients. MTT assay was applied to determine the proliferation and chemoresistance of A549 cells. Quantative PCR was used to detect the mRNA changes of Bcl-xL. Western blotting was used to detect the protein expression of Bcl-xL. ELISA was applied to detect the SDF-1 secretion from normal fibroblasts (NF) and CAF. CAF promoted the proliferation of A549 cells, while NF had no significant effect on them. After 72 hrs incubation, the absorbance value of A549+ CAF medium group was 0.814±0.006, significantly different from the 0.753±0.006 of the A549+ NF medium group (<0.05). The Q-PCR assay indicated that mRNA expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.11, 1.10±0.09 and 3.50±0.30, respectively, showing a significant difference between the A549+ NF medium group and A549+ CAF medium group (<0.05). The Western blot showed that protein expressions of Bcl-xL in the A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.08, 1.10±0.12 and 3.10±0.25, respectively, with a significant difference between the A549+ NF medium group and A549+ CAF medium group (<0.05). The ELISA results showed that the SDF-1 concentrations in the A549+ NF medium group and A549+ CAF medium group were 3.23±0.02 and 9.53±0.10, respectively, significantly different from each other (<0.05). The MTT assay indicated that the absorbance values of OD of A549 group, A549+ AMD3100 group, A549+ NF medium group, A549+ NF medium+ AMD3100 group, A549+ CAF medium and A549+ CA Fmedium+ AMD3100 group were 0.43±0.03, 0.25±0.02, 0.48±0.03, 0.31±0.03, 0.72±0.06 and 0.45±0.03, respectively. The data of A549+ NF medium group was significantly different from that of A549+ CAF medium group (<0.05). Cancer-associated-fibroblasts enhance the drug resistance of A549 cells through SDF-1 secretion, upregulating the expression level of Bcl-xL through interaction with CXCR4. Our study not only illustrates that tumor microenvironment is able to enhance drug resistance of tumor, but also provides experimental evidence for the cancer-associated-fibroblasts as a potential therapeutic target for the treatment of lung cancer.
为研究肿瘤微环境的关键组成部分——癌症相关成纤维细胞（CAF）是否通过分泌基质细胞衍生因子-1（SDF-1）来调节肺癌细胞系A549的化疗耐药能力。采用原代细胞分离技术从肺癌患者中分离癌症相关成纤维细胞。运用MTT法检测A549细胞的增殖和化疗耐药性。采用定量PCR检测Bcl-xL的mRNA变化。运用蛋白质免疫印迹法检测Bcl-xL的蛋白表达。采用酶联免疫吸附测定法检测正常成纤维细胞（NF）和CAF分泌的SDF-1。CAF促进A549细胞增殖，而NF对其无显著影响。孵育72小时后，A549+CAF培养基组的吸光度值为0.814±0.006显著不同于A549+NF培养基组的0.753±0.006（P<0.05）。定量PCR检测显示，A549组、A549+NF培养基组和A549+CAF培养基组中Bcl-xL的mRNA表达分别为1.00±u003cspan style="font-size:10.5pt;font-family:宋体">0.1u003cspan style="font-size:10.5pt;font-family:宋体">1、1.10±0.09和3.50±0.30，A549+NF培养基组与A549+CAF培养基组之间差异有统计学意义（P<0.05）。蛋白质免疫印迹法结果显示，A549组、A549+NF培养基组和A549+CAF培养基组中Bcl-xL的蛋白表达分别为1.00±0.08、1.10±0.12和3.10±0.25，A549+NF培养基组与A549+CAF培养基组之间差异有统计学意义（P<0.05）。酶联免疫吸附测定法结果显示，A�49+NF培养基组和A549+CAF培养基组中SDF-1浓度分别为3.23±0.02和9.53±0.10，差异有统计学意义（P<0.05）。MTT法检测显示，A549组、A549+AMD3100组、A549+NF培养基组、A549+NF培养基+AMD3100组、A549+CAF培养基组和A549+CAF培养基+AMD3100组的光密度（OD）吸光度值分别为0.43±0.03、0.25±0.02、0.48±0.03、0.31±0.03、0.72±0.06和0.45±0.03。A549+NF培养基组的数据与A549+CAF培养基组的数据差异有统计学意义（P<0.05）。癌症相关成纤维细胞通过分泌SDF-1增强A549细胞的耐药性，通过与CXCR4相互作用上调Bcl-xL的表达水平。我们的研究不仅表明肿瘤微环境能够增强肿瘤的耐药性，而且为癌症相关成纤维细胞作为肺癌治疗的潜在治疗靶点提供了实验依据。
