毛细管电泳免疫-激光诱导荧光术测定甲氨蝶呤对映体耐药A549细胞株中叶酰聚谷氨酸合成酶

Detection of folylpolyglutamate synthetase in methotrexate enantiomer induced resistant A549 cells by capillary electrophoresis-laser-induced fluorescence

目的为观察甲氨蝶呤（MTX）对映体诱导肺癌A549细胞株耐药后叶酰聚谷氨酸合成酶（folylpolyglutamate synthetase，FPGS）含量变化，建立一种用毛细管电泳免疫-激光诱导荧光术（CEIA-LIF）测定F＇PGS的方法，以便为深入探讨肿瘤耐药机制提供新的实验手段。方法实验分别选用耐药浓度为25μmol／L的L-（＋）-MTX和D-（-）-MTX肺癌A549耐药细胞株，以MTX敏感的细胞株作对照，培养获取上述3株细胞，并粗提取FPGS做CEIA-LIF和免疫印迹（WB）实验；用异硫氰酸荧光素（FITC）标记FPGS抗体，与提取的FPGS进行免疫反应；然后采用CEIA-LIF，根据不同分子量蛋白具有不同的迁移时间，分离检测标记蛋白，检测L-（＋）-MTX和D-（-）-MTX作用耐药前后3株肺癌A549细胞株中FPGS表达含量；同时用WB作对照，以评价CEIA-LIF的特异性和FPGS定量的准确性。结果CEIA-LIF分离标记FPGs抗体与免疫复合物的分离时间分别为7.1、8．9min，分离度（R）=4．5，在10min内即完成蛋白的分离和检测。经WB鉴定3株细胞液氮冻融裂解后离心提取液中组分与FIGS抗体无非特异性条带出现。CEIA-LIF测定敏感细胞株的检测下限为0．68mg／山细胞；而其检测25μmol／LL-（＋）-MTX和25μmol／LD-（-）-MTX诱导耐药细胞中FPGS的表达含量分别为对照组敏感细胞的46．59％和48．36％。结论本研究建立的CEIA-LIF检测FPGS法具有高效快速的特点，且具与WB类似的特异性；同时提高了检测的敏感度。L-（＋）-MTX和D-（-）-MTX诱导耐药后细胞株中FPGS表达含量较敏感细胞株明显减少，证明MTX耐药细胞株FPGS表达含量受损。

Objective To establish a method for detection folylpolyglutamate synthetase （FPGS）, explore the change of FPGS in the drug-resistant A549 ceils induced by methotrexate（MTX） enantiomer, and provide new tools to further investigate drug resistant mechanism. Methods A549 ceil lines induced by L-（＋）-MTX and D-（-）-MTX （25 μmo]/L） were chosen to raise three ceil lines as compared with MTX-sensitive ceil line. Then FPGS were extracted for the CEIA-LIF and western blot was performed. After validation, FPGS antibodies were labeled by fluorescein isothiocyanate （FITC） and produced a immune response with former-extracted FPGS. CELA-LIF can separate and detect labeled proteins according migration time of the protein with different size and detect FPGS in drug resistant ceil lines induced with L-（＋）-MTX and D-（-）-MTX. The accuracy was evaluated as compared with western blot assay. Results The separation time of CEIA-LIF for labeled FPGS antibody and the immune complexes were 7.1 rain and 8. 9 rain, and the resolving power was 4. 5. The process of protein separation and detection can be accomplished in less than 10 minutes. Western blot analysis showed there was no non-specific bands appears in the extract of these three ceil lines after the freeze-thaw in liquid nitrogen. The minimum detection level in sensitive ceil strains was 0. 68 mg/μL The consent of FPGS in L-（＋）-MTX and D-（-） -MTX induced cells were 46. 59% and 48.36% compared with drug sensitive cell strains with CELA-LIF. Conclusions CELA-LIF was established in this experiment. It is efficient and sensitive for detecting of FPGS, which is similar to western blot method. The level of FPGS in L-（＋）-MTX and D-（-）-MTX induced drug resistant cell lines is significantly lower, indicating the expression of FPGS is damaged.