多药耐药相关蛋白反义RNA对耐阿霉素人小细胞肺癌细胞株GLC4/ADR耐药性的调节

目的 构建表达多药耐药相关蛋白（MRP）反义RNA的真核表达载体，转染耐药细胞株，初步探讨其影响多药耐药（MDR）的作用机理。方法 以MRPcDNA为模板，应用PCR扩增MRPmRNA5′端（含起始子密码）及MRP mNRA3′端（含终止子密码）片段，通过定向克隆方法，构建表达MRP反义RNA的两个重组载体。通过Lipofectamine将其导入小细胞肺癌（SCLC）耐阿霉素（ADR）的细胞株GLC4／ADR中，经G418筛选，得到分别含pcDNA3空载（MO）、MRP mRNA5′端为靶区的反义RNA（Ma),MRPmRNA3′端为靶区的反义RNA（Mb)的3个细胞克隆。检测细胞内ADR的蓄积改变以及荷瘤裸鼠对ADR的敏感性。结果 经RT－PCR检测，外源片段可获稳定表达。Ma细胞及Mb细胞中，MRP表达分别抑制约14．0％和83．0％，且胞内ADR蓄积量均有一定程度的增加；对ADR的耐药倍数与对照细胞MO相比，分别下降9．5％和28．4％。虽然，MRP反义RNA对细胞的生长、细胞周期及ADR诱导的早期凋亡均无明显影响，但可提高荷瘤裸鼠对ADR的敏感性。结论 MRP反义RNA能抑制MRP蛋白的表达，并使转染细胞内ADR浓度增加，从而逆转MRP介导的MDR。对于配合化疗，提高MRP高表达患者的疗效，具有潜在的应用意义。

Modulation of human small cell lung cancer cell line GLC4/ADR multidrug resistance in the inhibition of multidrug resistance-associated protein and its antisense

OBJECTIVE: To study the effect of antisense multidrug resistance-associated protein (MRP) RNA on multidrug resistance (MDR) in the human small cell lung cancer (SCLC) cell line GLC4/ADR, in which the overexpression of MRP gene is discovered. METHODS: In using the plasmid pRC/RSV-MRP1 containing complete ORF of MRP as a template, two antisense recombinants targeting at the 5' and 3' regions were constructed with the application of the polymerase chain reaction (PCR) technique. Lipofectamine was conducted to transduce these antisense MRPs into the GLC4/ADR cells. Three clones (the GLC4/ADR-pcDNA3, MO; GLC4/ADR-MRP-5' region, Ma; GLC4/ADR-MRP-3' region, Mb) of transfectants after the selection of G418 were obtained. The expression of MRP protein was detected by the use of the Western blot and the MTT method for determination of chemosensitivity to ADR was used. RESULTS: The antisense MRPs was found to be effectively expressed in the clones, being transfected with two different antisense MRPs. The MRP expression in these transfectants were inhibited at the rates of 14.0% (GLC4/ADR Ma) and 83.0% (GLC4/ADR Mb), respectively. Moreover, decrease in the ADR resistance was observed in the Ma and Mb at the rates of 9.5% and 28.4%. Although the intracellular ADR concentration was increased in these transfectants, the proliferation and cell cycle and their early apoptosis induced by the ADR, indicated no difference between the transfectants and parental cells. CONCLUSION: It is possible to obtain the effective expression of the MRP antisense structures for the blocking of the mRNA translation in GLC4/ADR cell line and the fragment complementary to the 3'-region of MRP is more effective for the inhibition of the MRP than that related to the 5'-region of MRP. The antisense RNA may be a useful treatment in combination with the conventional chemotherapy for SCLC, in which the MRP overexpression usually occurs.