稳定过表达成纤维细胞激活蛋白的口腔鳞状细胞癌细胞株的构建

目的 构建人成纤维细胞激活蛋白（FAP）过表达慢病毒载体,转染口腔鳞状细胞癌（OSCC）细胞株SCC9,构建稳定过表达FAP的OSCC细胞株。方法 采用聚合酶链反应（PCR）技术获得FAP片段,利用基因重组技术构建过表达FAP的慢病毒载体,包埋病毒并收集上清液感染SCC9细胞株,通过流式细胞荧光分选技术（FACS）进行筛选,获得稳定过表达FAP的SCC9细胞株。结果 对阳性克隆进行酶切鉴定和基因测序证明FAP的慢病毒载体构建成功,实时荧光定量PCR和Western blot检测结果证明成功构建稳定过表达FAP的SCC9细胞株。结论 本研究有助于获得纯化FAP蛋白,为进一步研究FAP在OSCC发生发展过程中的机制奠定基础。

Construction of a eukaryotic expression vector of fibroblast activation protein and establishment of its stable over- expression in th eoral squamous cell carcinoma

Objective This study aimed at constructing fibroblast activation protein (FAP) over-expression lentivinus vectors to investigate transfection in SCC9 cell lines and establish a stable FAP over-expression oral squamous cell line. Methods The cDNA of FAP gene from an oral squamous cell carcinoma (OSCC) tissue was amplified by polymerase chain reaction (PCR) and subcloned into eukaryotic expression vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid was sequenced and then transfected into an SCC9 cell line. Subsequently, the SCC9 cell line that over-expressed FAP stably was established by fluorescence activated cell sorting (FACS). The expression of green fluorescent protein (GFP) was detected with fluorescence microscopy, and the over-expression of FAP was identified by real-time PCR and Western blot.Results The FAP gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. GFP was expressed in the transfected cells. Furthermore, FAP over-expression in the transfected cells was detected by real-time PCR and Western blot.Conclusion The recombinant eukaryotic expression vector pCDH-FAP was constructed success-fully. This result provides a foundation for further studies on the function of FAPin vitro.