CAJ | 학술논문

目的构建在人肝癌HepG2细胞内可稳定表达血管内皮生长因子受体3(VEGFR3)的逆转录病毒载体。方法采用PCR技术扩增VEGFR3片段，克隆至pBABE-puro质粒，构建重组质粒pBABE-puro-VEGFR3，经PCR﹑酶切﹑测序等验证后，体外与PIK包装质粒共同转染293FT细胞，获得携带VEGFR3基因的重组逆转录病毒，感染人肝癌HepG2细胞，获得转入VEGFR3的HepG2细胞，通过QPCR和Western blot进一步验证VEGFR3在HepG2细胞内的表达。结果①获得携带VEGFR3基因的重组质粒pBABE-puro-VEGFR3和人肝癌细胞株HepG2-pBABE-puro-VEGFR3；②QPCR和Western blot分别检测到VEGFR3在靶细胞HepG2-pBABE-puro-VEGFR3中的稳定表达。结论成功构建了携带VEGFR3基因的人肝癌细胞重组逆转录病毒载体。
목적구건재인간암HepG2세포내가은정표체혈관내피생장인자수체3(VEGFR3)적역전록병독재체。방법채용PCR기술확증VEGFR3편단，극륭지pBABE-puro질립，구건중조질립pBABE-puro-VEGFR3，경PCR﹑매절﹑측서등험증후，체외여PIK포장질립공동전염293FT세포，획득휴대VEGFR3기인적중조역전록병독，감염인간암HepG2세포，획득전입VEGFR3적HepG2세포，통과QPCR화Western blot진일보험증VEGFR3재HepG2세포내적표체。결과①획득휴대VEGFR3기인적중조질립pBABE-puro-VEGFR3화인간암세포주HepG2-pBABE-puro-VEGFR3；②QPCR화Western blot분별검측도VEGFR3재파세포HepG2-pBABE-puro-VEGFR3중적은정표체。결론성공구건료휴대VEGFR3기인적인간암세포중조역전록병독재체。
Objective To construct a retroviral vector carrying vascular endothelial growth factor receptor-3(VEGFR3) and transfect VEGFR3 into HepG2 cells. Methods VEGFR3 gene was amplified by PCR and subcloned into the retroviral vector pBABE-puro to construct a retroviral plasmid (pBABE-puro-VEGFR3) expressing VEGFR3. The VEGFR3 gene insert was tested by PCR, restriction enzyme digestion, and DNA sequencing. The recombinant retroviruses carrying VEGFR3 were produced in 293FT cells co-transfected with pBABE-puro-VEGFR3 and the packaging plasmids PIK, and then infected HepG2 cells. The VEGFR3 mRNA and protein expressions were determined by the reverse transcription-PCR and Western blotting, respectively. Results The retroviral plasmid carrying VEGFR3(pBABE-puro-VEGFR3) was constructed successful y. The expressions of VEGFR3 mRNA and VEGFR3 protein in hepG2 cells after delivered VEGFR3 into pBABE-puro-VEGFR3 could be detected by QPCR and Western blot. Conclusion The recombinant retrovirus vector hepG2-pBABE-puro-VEGFR3 has been successful y constructed, which can stable express VEGFR3 protein.