持续高表达CXC趋化因子受体-4重组慢病毒颗粒的包装鉴定及细胞迁移  

Sustained high expression of CXC chemokine receptor-4 recombinant lentiviral particles identifica-tion and cell migration

作　　者：王奇[1] 冯世庆[1] 孔晓红[1] 刘畅[1] 张彬[1] 张亮[1] 张衍军[1] 

机构地区：[1]天津医科大学总医院骨科,天津300052

出　　处：《中华实验外科杂志》2013年第3期605-608,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81070982）;天津市应用基础及前沿技术研究计划重点项目（IOJCZDJCl8800）

摘　　要：目的观察持续高表达CXC趋化因子受体_4（CXCR4）慢病毒颗粒在细胞迁移的作用。方法从大鼠大脑组织提取总RNA，逆转录一聚合酶链反应（RT—PCR）得到大鼠cDNA文库，以cDNA为模板，经过PCR获得CXCR4基因，测序正确后连接到pCDHl一MCS—EFl一copGFP（慢病毒载体质粒），与第3代慢病毒其余辅助质粒pMDLG／pRRE、pRsv．Rev及pVSV．G按照1：4：1：1共转染293T细胞包装慢病毒，超离慢病毒，测定病毒滴度，感染Hela细胞72h与14d后，分别观察Hela细胞荧光，实时定量聚合酶链反应（Real—timepcn）测定Hela细胞表达大鼠CXCR4mRNA含量，Transwell小室观察细胞迁移。结果测序CXCR4基因序列正确，并成功连接到pCDHl．MCS—EFl．copGFP，利用第3代慢病毒包装系统成功包装慢病毒，经超离慢病毒颗粒，流式细胞仪测定慢病毒滴度为7．89×10^5，持续观察Hela细胞荧光强度，Real．timePCR检测结果示Hela细胞表达大鼠CXCR4mRNA水平显著升高（72h为78．29倍，至14d为58．93倍），Transwell小室观察到高表达CXCR4的慢病毒颗粒感染组细胞迁移多（P〈0．05）。结论高效感染力并持续高表达CXCR4的慢病毒颗粒可持续促进细胞迁移，并有基质细胞衍生因子．1d（SDF一10L）浓度依赖性。Objective To explore the sustained high expression of CXC chemokine receptor-4 （CXCR4） lentiviral particles in cell migration.＇Methods Total RNA was extracted from rat brain tissues. The rat cDNA library was obtained by using reverse transcription-polymerase chain reaction （RT-PCR）. CXCR4 gene was acquired through PCR with the eDNA library as template. After CXCR4 gene correct se- quencing, it was linked to pCDH1-MCS-EFI-copGFP lentiviral vector plasmid, and co-transfected with the rest of the third generation lentiviral helper plasmid pMDLG/pRRE, pRsv-Rev, and pVSV-G into 293T cells in the ratios of 1： 4： 1：1 for packaging lentiviral particles. After infection for 48 h, supernatant was collected, and subjected to super centrifugation of lentivirus. The lentivirus titer was determined by using flow eytometry. After infection of Hela cells for 72 h and 14 days, the fluorescence in Hela cells was ob- served. The coritent of rat CXCR4 mRNA in Hela cells was detected by using real-time PCR, and the mi- gration of Hela cells was observed by using Transwell chamber. Results The CXCR4 gene sequence was correct, and successfully linked to the pCDH1-MCS-EFI-copGFP lentiviral vector plasmid. The third gen- eration lentivirus particles were successfully packaged, and the lentivirus particles were super-centrifuged. The lentivirus titer was 7. 89 x 10^5 measured by using flow eytometry： The fluorescence intensity of Hela cells was sustained strong. In Hela cells the expression levels of rat CXCR4 mRNA were significantly ele- vated （72 h： 78. 29, and 14 days： 58. 93 times）. Hela cells infected with high expression of CXCR4 lenti- viral particles migrated more than the other groups （ P 〈 0. 05 ）. Conclusion The lentiviral particles with efficient infection and sustained high expression of CXCR4 can continuously promote cell migration stromal cell derived factor （SDF） -1 a-concentration-dependently.

关 键 词：CXC趋化因子受体-4 基质细胞衍生因子-1a 慢病毒 细胞迁移 

分 类 号：R[医药卫生]