摘要
目的:构建人紫杉醇耐药基因1(TXR1)的表达载体并对MCF-7人乳腺癌细胞系进行转染且检测其表达。方法:RT-PCR方法扩增TXR1 CDS片段,并将扩增的片段插入pEGFP-C3真核表达载体,构建成含TXR1 cDNA的重组载体pEGFP-TXR1,以脂质体介导该质粒转染人乳腺癌细胞系MCF-7,应用RT-PCR、Western Blot和荧光显微镜鉴定转染细胞中TXR1的表达。用MTT法对转染前后的MCF-7细胞进行紫杉醇耐药性分析。结果:成功构建含TXR1 cDNA的重组载体pEGFP-TXR1,转染MCF-7细胞后成功表达TXR1蛋白,在转染的细胞中,证实有TXR1 mRNA和蛋白表达上调。MTT法显示转染了TXR1的MCF-7细胞获得紫杉醇耐药性。结论:成功构建TXR1表达载体,并在人乳腺癌细胞系MCF-7得到表达,获得紫杉醇耐药性。
Objective： To construct a TXR1 expression vector and study the expression of the protein in a human breast cancer cell line. Methods： TXR1 cDNA was amplified by reverse transcription-polymerase chain reaction （RT-PCR） and was then inserted into pEGFP-C3 vector to construct pEGFP-TXRI. After confirmation by DNA sequencing, the recombinant pEGFP-TXR1 was transfected into cultured MCF-7 cells by lipofectamine 2000. The expression of TXR1 in transfected cells was assessed by RT-PCR and immunoblotting, and MTT assay was used to detect cell proliferation and construct an inhibition curve for Taxol. Results： TXR1 expression was highly expressed in MCF-7 cells containing the recombi- nant expression vector. The survival rate of MCF-7 cells with high expression of TXR1 increased. Conclusion： A recombinant TXR1 expression vector has been successfully constructed. It expresses TXR1 in MCF-7 breast cancer cells and cells with a high expression level of TXR1 acquire Taxol resistance.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2010年第21期1205-1208,共4页
Chinese Journal of Clinical Oncology
关键词
TXR1
质粒构建
乳腺癌细胞
紫杉醇
化疗耐药
TXR1 protein
Plasmid construction
Breast cancer cell
Paclitaxel
Drug resistance
