title: [Effect of CXCR4 gene overexpression mediated by lentiviral vector on the biological characteristics of mesenchymal stem cells].
abstract: OBJECTIVE
  To construct mouse CXC chemokine receptor type 4 (Cxcr4) gene overexpressing lentiviral vector and to evaluate its biological effect on mouse mesenchymal stem cells (MSCs).
  
  
  METHODS
  Cxcr4 gene was amplified and subcloned into pCR-Blunt vector. Cxcr4 gene and enhanced green fluorescent protein (EGFP) gene expressed bicistronic recombinant lentiviral vector LV-CXCR4-IRES-EGFP and control vector LV-IRES-EGFP were constructed, respectively. Both plasmids were co-transfected into 293FT packaging cell line with packaging plasmid pSPAX2 and enveloping plasmid pMD.2G using Lipofectamine 2000 to produce lentiviral virus, respectively. The recombinant viruses were harvested and the virus titer was determined by limiting dilution. Mouse MSCs were infected with viral supernatant. EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM). Cell counting kit-8 (CCK-8) was applied in mixed lymphocyte reaction (MLR) to evaluate the suppressive effect of MSCs on mice splenocyte proliferation in vitro. Wound healing ability of MSCs was measured by scratch experiment and migration capacity by a chemotaxis assay using a transwell assay.
  
  
  RESULTS
  The Cxcr4 fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) and verified by DNA sequencing. The restriction enzyme digestion experiment demonstrated that the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and the control vector LV-IRES-EGFP were successfully constructed. Expression of CXCR4 was detected by fluorescence microscopy, which indicated that the lentiviral particles expressing CXCR4 were packaged. Furthermore, expression of EGFP were detected by fluorescence microscopy in MSCs after infection and the expression of CXCR4 protein on MSCs surface in CXCR4-MSC group was significantly increased comparing to those in the control group(P < 0.05).CXCR4-MSCs group and the control group were (90.3 ± 3.37)% and (1.53 ± 0.34)%, respectively. Meanwhile, overexpression of CXCR4 had no effect on their capacity of immune regulation when co-cultured with splenocyte(P > 0.05). Moreover, overexpression of CXCR4 can not only accelerated the wound healing after scratch, but also enhanced the migration ability of cells in the transwell induced by high concentration of SDF-1 in a dose-dependent manner compared with the EGFP control group.
  
  
  CONCLUSIONS
  The CXCR4 expressing lentiviral vector LV-CXCR4-IRES-EGFP was successfully constructed. The lentiviral vector can not only efficiently infect mouse MSCs, but also stably express CXCR4 in MSCs. The MSCs modified with CXCR4 have biological characteristic in vitro.
authors.affiliation: Department of Hematology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Miao
authors.initials: M
authors.lastname: Li
authors.forename: Zhi-ling
authors.initials: ZL
authors.lastname: Yan
authors.forename: Hai
authors.initials: H
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Pan
authors.forename: Ling-yu
authors.initials: LY
authors.lastname: Zeng
authors.forename: Zhen-yu
authors.initials: ZY
authors.lastname: Li
authors.forename: Kai-lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D019718:Receptors, CXCR4 / Q000235:genetics*
chemical_list: D019718:Receptors, CXCR4