通过基因合成的TPL2(tumor progression locus 2)质粒,获得稳定表达TPL2的PK-15/TPL2细胞系。将慢病毒载体Lv-PCDH和TPL2(Pig)重组慢病毒载体Lv-TPL2(Pig)利用转染试剂将其转染至PK-15细胞。感染48 h后,加入嘌呤霉素进行药物筛选,通过...通过基因合成的TPL2(tumor progression locus 2)质粒,获得稳定表达TPL2的PK-15/TPL2细胞系。将慢病毒载体Lv-PCDH和TPL2(Pig)重组慢病毒载体Lv-TPL2(Pig)利用转染试剂将其转染至PK-15细胞。感染48 h后,加入嘌呤霉素进行药物筛选,通过筛选得到的阳性细胞株PK-15/TPL2即为稳定表达TPL2蛋白的PK-15细胞株。通过qRT-PCR法和琼脂糖凝胶电泳、IFA,TCID50技术验证PK-15/TPL2细胞系和对口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪塞内加谷病毒(seneca valley virus,SVV)病原复制的影响。结果显示,细胞表达大量的TPL2的基因产物,用FMDV、SVV病毒感染稳定表达TPL2的PK-15细胞株时,明显抑制病毒的复制。结果表明,利用该技术可高效快速地建立了稳定高表达TPL2的PK-15细胞系,该细胞株的建立为病毒的分离及TPL2的生物学活性研究奠定理论基础。展开更多
Overexpression of breast cancer resistance protein （ABCG2/BCRP） in cancer cells may cause tumor resistance to chemotherapeutic drugs. RNA interference （RNAi） can selectively silence the expression of a target gene...Overexpression of breast cancer resistance protein （ABCG2/BCRP） in cancer cells may cause tumor resistance to chemotherapeutic drugs. RNA interference （RNAi） can selectively silence the expression of a target gene of interest. In the present study, we aimed to modulate the BCRP expression and examine the functional consequence using RNAi approach. Three siRNAs （si-BCRP1, si-BCRP2 and si-BCRP3） targeting BCRP were evaluated in drug-resistant MCF-7/MX100 cells overexpressing BCRP. The BCRP expression at the mRNA and protein levels was inhibited by si-BCRP2 and si-BCRP3 over 90% and 70%, respectively. As a result, the intracellular mitoxantrone accumulation was sharply increased in MCF-7/ MX100 cells after the transfection. Furthermore, shRNA sequences bearing si-BCRP2 and siBCRP3 were cloned into lentiviral expression plasmid （pTRIPZ） to package lentivirus, and MCF-7/MX100 cells stably expressing siRNA targeted to human ABCG2/BCRP were established by lentivector-mediated gene transfer system. The stable cells exhibited an increased miotxantrone accumulation, among which the BCRP expression at the mRNA level was reduced by Lenti-BCRP2 and Lenti-BCRP3 around 72% and 56%, respectively. Moreover, the BCRP expression at the protein level was reduced by 70% and 53%, respectively. Furthermore, the cell lines were used to screen active ingredients in traditional herbal medicines in order to evaluate BCRP substrates or inhibitors. Our data suggested that the BCRP knockdown cell lines could serve as good cell models for preclinical studies.展开更多
文摘通过基因合成的TPL2(tumor progression locus 2)质粒,获得稳定表达TPL2的PK-15/TPL2细胞系。将慢病毒载体Lv-PCDH和TPL2(Pig)重组慢病毒载体Lv-TPL2(Pig)利用转染试剂将其转染至PK-15细胞。感染48 h后,加入嘌呤霉素进行药物筛选,通过筛选得到的阳性细胞株PK-15/TPL2即为稳定表达TPL2蛋白的PK-15细胞株。通过qRT-PCR法和琼脂糖凝胶电泳、IFA,TCID50技术验证PK-15/TPL2细胞系和对口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪塞内加谷病毒(seneca valley virus,SVV)病原复制的影响。结果显示,细胞表达大量的TPL2的基因产物,用FMDV、SVV病毒感染稳定表达TPL2的PK-15细胞株时,明显抑制病毒的复制。结果表明,利用该技术可高效快速地建立了稳定高表达TPL2的PK-15细胞系,该细胞株的建立为病毒的分离及TPL2的生物学活性研究奠定理论基础。
基金supported by the National Natural Science Foundation of China (No. 30240011)the Key Technologies Program of the Scientific and Technical Bureau of Hebei Province China (No. 2007000858)
基金National Major Projects of the Ministry of Science and Technology of China (Grant No.2012ZX09506001-004)
文摘Overexpression of breast cancer resistance protein （ABCG2/BCRP） in cancer cells may cause tumor resistance to chemotherapeutic drugs. RNA interference （RNAi） can selectively silence the expression of a target gene of interest. In the present study, we aimed to modulate the BCRP expression and examine the functional consequence using RNAi approach. Three siRNAs （si-BCRP1, si-BCRP2 and si-BCRP3） targeting BCRP were evaluated in drug-resistant MCF-7/MX100 cells overexpressing BCRP. The BCRP expression at the mRNA and protein levels was inhibited by si-BCRP2 and si-BCRP3 over 90% and 70%, respectively. As a result, the intracellular mitoxantrone accumulation was sharply increased in MCF-7/ MX100 cells after the transfection. Furthermore, shRNA sequences bearing si-BCRP2 and siBCRP3 were cloned into lentiviral expression plasmid （pTRIPZ） to package lentivirus, and MCF-7/MX100 cells stably expressing siRNA targeted to human ABCG2/BCRP were established by lentivector-mediated gene transfer system. The stable cells exhibited an increased miotxantrone accumulation, among which the BCRP expression at the mRNA level was reduced by Lenti-BCRP2 and Lenti-BCRP3 around 72% and 56%, respectively. Moreover, the BCRP expression at the protein level was reduced by 70% and 53%, respectively. Furthermore, the cell lines were used to screen active ingredients in traditional herbal medicines in order to evaluate BCRP substrates or inhibitors. Our data suggested that the BCRP knockdown cell lines could serve as good cell models for preclinical studies.
