CAJ | 학술논문

目的构建带有荧光标记基因增强型绿色荧光蛋白（EGFP）和apelin基因的重组质粒pUbi-apelin-FLAG-pSV40-EGFP并进行慢病毒包装，探讨其转染人脐带间充质干细胞的最佳感染复数（MOI）值及目的基因表达情况。方法化学合成目的基因片段并扩增。采用In-Fusion技术将酶切后的目的基因片段与线性质粒载体连接，转化入感受态DH5α细胞中后筛选阳性克隆并进行测序。重组质粒慢病毒载体转染293T细胞，包装慢病毒并测定滴度。将重组质粒慢病毒按不同MOI值转染人脐带间充质干细胞，根据转染效率得到最佳MOI值，并采用Real-time-PCR及Western blot方法检测目的基因表达情况。结果通过PCR扩增获得酶切位点碱基修饰后的大小约284 bp的目的基因片段，与慢病毒质粒载体连接后形成pUbi-apelin-FLAG-pSV40-EGFP重组质粒，测序结果与预期完全符合，并成功包装慢病毒颗粒。最佳MOI值为20，转染效率为（90.32±3.61）%。慢病毒载体能高效转染人脐带间充质干细胞且2周内持续稳定上调目的基因mRNA及蛋白的表达。结论重组质粒慢病毒载体pUbi-apelin-FLAG-pSV40-EGFP可有效转染人脐带间充质干细胞，并可持续高表达apelin基因。
목적구건대유형광표기기인증강형록색형광단백（EGFP）화apelin기인적중조질립pUbi-apelin-FLAG-pSV40-EGFP병진행만병독포장，탐토기전염인제대간충질간세포적최가감염복수（MOI）치급목적기인표체정황。방법화학합성목적기인편단병확증。채용In-Fusion기술장매절후적목적기인편단여선성질립재체련접，전화입감수태DH5α세포중후사선양성극륭병진행측서。중조질립만병독재체전염293T세포，포장만병독병측정적도。장중조질립만병독안불동MOI치전염인제대간충질간세포，근거전염효솔득도최가MOI치，병채용Real-time-PCR급Western blot방법검측목적기인표체정황。결과통과PCR확증획득매절위점감기수식후적대소약284 bp적목적기인편단，여만병독질립재체련접후형성pUbi-apelin-FLAG-pSV40-EGFP중조질립，측서결과여예기완전부합，병성공포장만병독과립。최가MOI치위20，전염효솔위（90.32±3.61）%。만병독재체능고효전염인제대간충질간세포차2주내지속은정상조목적기인mRNA급단백적표체。결론중조질립만병독재체pUbi-apelin-FLAG-pSV40-EGFP가유효전염인제대간충질간세포，병가지속고표체apelin기인。
Objective To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with lentivirus to co-express enhanced green fluorescent protein (EGFP) and apelin, and to investigate optimal multiple of infec-tion (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene. Methods The apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into linear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme di-gestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The trans-fection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and Western blot assay. Results A 284 bp target gene fragment with the restriction sites was obtained by PCR and connected to the lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentivi-ral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The mRNA and protein levels of target gene were stably up-regulated within 2 weeks. Conclusion The lentivirus vector pUbi-apelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express high levels of apelin gene in two weeks.