Objective: 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. Methods: 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. Results: 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 (P<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 (P<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 (P<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 (P<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 (P<0.05). Conclusions: 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.
目的： 探讨肿瘤相关成纤维细胞(CAF)是否通过分泌基质细胞衍生因子1(SDF-1)调节肺癌A549细胞的化疗耐药性。 方法： 采用原代细胞分离方法获得肺癌患者的CAF，采用四甲基偶氮唑蓝(MTT)法检测A549细胞的增殖能力和对化疗药物的耐药性，荧光定量PCR检测Bcl-xL mRNA水平，Western blot检测Bcl-xL蛋白水平，酶联免疫吸附试验(ELISA)检测CAF分泌SDF-1的情况。 结果： CAF可以促进肺癌A549细胞的增殖，而正常纤维细胞对A549细胞增殖的影响不明显。培养72 h后，A549＋CAF上清组和A549＋NF上清组的吸光度(A)值分别为0.814±0.006和0.753±0.006，差异有统计学意义(P<0.05)。荧光定量PCR检测结果显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL mRNA表达水平分别为1.00±0.11、1.10±0.09和3.50±0.30，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。Western blot检测显示，A549组、A549＋NF上清组和A549＋CAF上清组的Bcl-xL蛋白表达水平分别为1.00±0.08、1.10±0.12和3.10±0.25，A549＋CAF上清组与A549＋NF上清组比较，差异有统计学意义(P<0.05)。ELISA检测显示，A549＋NF上清组和A549＋CAF上清组中SDF-1含量分别为3.23±0.02和9.53±0.10，差异有统计学意义(P<0.05)。MTT检测显示，A549组、A549＋AMD3100组、A549＋NF上清组、A549＋NF上清＋AMD3100组、A549＋CAF上清组和A549＋CAF上清＋AMD3100组的A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＋CAF上清和A549＋NF上清组比较，差异有统计学意义(P<0.05)。 结论： CAF通过分泌SDF-1激活肺癌A549细胞中的趋化因子受体4(CXCR4)，提高Bcl-xL的表达，增强肺癌A549细胞对顺铂的化疗耐药性，这为CAF作为肺癌潜在的治疗靶点提供了实验依据。.
Keywords: Bcl-xL; Cancer-associated-fibroblasts; Drug resistance; Lung neoplasms; Stromal cell derived factor-1; Tumor microenvironment.