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目的体外建立人肺腺癌细胞多药耐药细胞系,观察其生物学特性并探讨其细胞内DNA polβ和多药耐药基因mdr1、mrp1、GST-πl、rp和topo Ⅱ的表达及意义。方法以紫杉醇为诱导药,采用间歇逐步增加剂量法建立人肺腺癌多药耐药细胞系A549/TXL20;MTT法检测A549/TXL20对紫杉醇、顺铂、长春新碱、5-氟尿嘧啶和丝裂霉素的耐药指数(RF);细胞克隆形成法测定A549/TXL20对紫杉醇的敏感性;高效液相色谱测定细胞内紫杉醇的浓度;RT-PCR法测定人肺腺癌多药耐药细胞系A549/TXL20中DNA polβ、mdr1、GST-π、mrp1l、rp和topo Ⅱ的基因表达。结果①A549/TXL20形态不规则,较亲本细胞略小,核/浆比增加,倍增时间没有明显变化;②A549/TXL20对紫杉醇的RF为19.3,对顺铂的RF为67.4,对长春新碱、5-氟尿嘧啶、丝裂霉素和顺铂等抗癌药物有不同程度的耐药性;③A549/TXL20对紫杉醇的敏感性降低,其紫杉醇的含量较亲本细胞下降;④A549/TXL20细胞中DNA polβ、mdr1、GST-π、mrp1l、rp的基因表达量分别较A549细胞中的表达量明显增加,差异具有统计学意义(P<0.05)。结论建立了相对稳定的人肺腺癌多药耐药细胞系A549/TXL20,其耐药机制可能与mdr1、GST-π、mrp1l、rp和DNA polβ基因的高表达有关。
Objective To establish a multidrug-resistant cell line of human lung adenocarcinoma cells in vitro and observe its biological characteristics and to explore the expression and significance of the intracellular polβ and multidrug resistance genes mdr1, mrp1, GST-πl, rp and topo Ⅱ. Methods Paclitaxel was used as an inducing agent to establish a multidrug-resistant human lung adenocarcinoma cell line A549 / TXL20 by intermittent increasing dose method. MTT assay was used to detect the inhibitory effect of A549 / TXL20 on paclitaxel, cisplatin, vincristine, 5-fluorouracil and mitosis (RF). A549 / TXL20 was assayed for its sensitivity to paclitaxel by cell clone formation assay. Intracellular paclitaxel concentration was determined by high performance liquid chromatography (RP-HPLC). The multidrug resistance of human lung adenocarcinoma cell line A549 / TXL20 DNA polβ, mdr1, GST-π, mrp1l, rp and topo Ⅱ gene expression. Results ① The morphology of A549 / TXL20 was irregular and slightly smaller than that of the parental cells. The nuclear / plasma ratio increased and the doubling time did not change significantly. The RF of A549 / TXL20 was 19.3 for paclitaxel and 67.4 for cisplatin, --Anti-chemosensitivity of anticancer drugs such as fluorouracil, mitomycin and cisplatin; ③The sensitivity of A549 / TXL20 to paclitaxel decreased, the content of paclitaxel decreased compared with that of parental cells; ④ The DNA polβ, mdr1 , Respectively. The gene expression of GST-π, mrp1l and rp in A549 cells was significantly higher than that in A549 cells, the difference was statistically significant (P <0.05). Conclusions A relatively stable human lung adenocarcinoma multidrug resistance cell line, A549 / TXL20, was established and its mechanism of drug resistance may be related to the overexpression of mdr1, GST-π, mrp1l, rp and DNA polβ genes.