| Aim：To construct lentiviral vector overexpressing mouse C-X-C chemokine receptortype4(Cxcr4) gene and to evaluate its effect on mouse mesenchymal stem cell. Toinvestigate the homing capacity of CXCR4overexpressed mesenchymal stem cells(MSC), and their effect on hematopoietic recovery, as well as the prevention andprotection on mouse graft-versus-host disease.Methods:(1) Cxcr4gene was amplification and subcloned into pCR-Blunt vector.The recombinant lentiviral vector LV-CXCR4-IRES-EGFP and LV-IRES-EGFP wasconstructed by digesting pCR-Blunt-Cxcr4and EGFP and then ligased into LV-lacZvector. Both plasmids were co-transfected into293FT packaging cell line withpackaging plasmid pSPAX2and envelope plasmid pMD.2G using lipofectamine2000to produce lentiviral virus, respectively. The recombinant viruses were harvested andthe virus titer was determined by limiting dilution.(2) Mouse MSCs were infectedwith viral supernatant. EGFP expression was visualized using fluorescencemicroscope and efficiency of infection was determined by flow cytometry (FCM).Healing ability of MSCs was measured by scratch and a chemotaxis assay performedusing a transwell assay. Cells were counted by hemocytometer to evaluate theproliferation. Cell apoptosis was detected by flow cytometer for7-amino-actinomycinD (7-AAD) and AnnexinⅤ. Cell counting kit-8(CCK-8) method was applied inmixed lymphocyte reaction (MLR) to evaluate the suppressive effect of MSCs onmice splenocyte proliferation in vitro.(3) BALB/c mice were lethally irradiated24hours before5×10~5CXCR4-MSC or EGFP-MSC were injected. Twenty four hoursafter transplantation, recipients were sacrificed; frozen sections were prepared to detect the distribution of infused MSCs. Furthermore, the number of MSCs homing tospleen and bone marrow was measured by flow cytometer. The level of SDF-1inrecipients’ serum and bone marrow was detected by ELISA.(4) Four hours after TBI,5×10~6bone marrow cells (BMC) from BALB/c mice were infused into BALB/crecipient mice to establish a syngeneic bone marrow transplantation model. Aftertwenty four hours, recipients were injected intravenously5×10~5CXCR4-MSC orEGFP-MSC. General status and survival rate was observed every day. Peripheralblood cells were counted by hemocytometer twice a week, and hematopoietic stemcell in bone marrow was detected by flow cytometer, as well as bone marrow andspleen was assessed by histopathological examination.(5) Four hours after TBI,5×10~6BMC from C57BL/6mice (H-2K~b) were infused into BALB/c recipient mice(H-2K~d), which established an allogeneic bone marrow transplantation model,additional2×106donor’s splenocytes were infused for establishing graft-versus-hostdisease (GVHD) model. After twenty-four hours,5×10~5CXCR4-MSC or EGFP-MSCwere injected through tail vein. Recipient mice were divided into5groups: TBI group,allo-BMT group, GVHD group, GVHD+EGFP-MSC group, and GVHD+CXCR4-MSC group. The recipients were monitored daily for general state andsurvival, and every two days for weight changes. GVHD was evaluated by clinicalsigns, histopathological changes and peripheral proinflammatory cytokines changes.Meanwhile, T helper cell subsets in peripheral blood were examined by flowcytometry. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ,TNF-α and IL-17A were determined using a Cytometric Bead Array (CBA) kit.Results:(1) The Cxcr4fragment was amplified by reverse transcription polymerasechain reaction (RT-PCR) and verified by DNA sequencing. The restriction enzymedigestion experiment demonstrated that the recombinant lentiviral vectorLV-CXCR4-IRES-EGFP and the control vector LV-IRES-EGFP was successfully constructed. Expression of EGFP was detected by fluorescence microscopy, whichindicated the lentiviral particles expressing CXCR4were packaged.(2) Furthermore，expression of EGFP were detected by fluorescence microscopy in MSCs afterinfection and the efficiency of infection reached70%after optimization of infectioncondition. Expression of CXCR4protein on MSCs surface in CXCR4-MSC groupwas significantly increased comparing to those in the control group. Moreover,CXCR4not only accelerated the wound healing after scratch, but also enhanced themigration of cells in the transwell induced by SDF-1gradient compared with theEGFP control group. Meanwhile, overexpressing of CXCR4had no effect on theproliferation and apoptosis of MSCs, as well as their capacity of immune regulationwhen co-cultured with splenocyte.(3)After twenty four hours transfusion, MSCswere observed in recipients’ liver, spleen and lung both in EGFP-MSC group andCXCR4-MSC group. Compared to the EGFP-MSC group, the number of MSCshoming to spleen and bone marrow increased in CXCR4-MSC group. Furthermore,the level of SDF-1in serum and bone marrow significantly increased after lethalirradiation.(4) All groups received syngeneic BMT acquired100%survival exceptTBI group. Generally, recovery of white blood cells and platelet in peripheral bloodwas earlier in mice infused with MSCs. Moreover, the percentage of KSL(c-kit~+Sca-1~+Lin~-) cells showed earlier hematopoietic recovery in CXCR4-MSCgroup than that of syn-BMT group. The histopathological examination of spleen andbone marrow showed faster hematopoietic recovery in CXCR4-MSC group thansyn-BMT group.(5) There were visible changes of survival rate among GVHDgroups. The survival rate in MSCs infused groups was higher than mice withoutMSCs infused. The status of consciousness, appetite, weight, and movement wererecovered earlier compared to mice without transplanted with MSCs. Hematopoieticrecovery in MSCs infused group was faster than GVHD group. Typical manifestation such as bowing, diarrhea was observed in GVHD group. While, the clinical score washigher than the mice which received transplants with CXCR4-MSC and EGFP-MSC.There was significant difference between the CXCR4-MSC group and GVHD group（P<0.05）. Compared to GVHD group, mice with CXCR4-MSC infused promotedimmune reconstitution, including early restoration of bone marrow microenvironmentand changes of immunocyte in peripheral blood. Furthermore, in CXCR4-MSC group,T lymphocytes including CD4~+T cells had earlier recovery, recovery of Blymphocytes and NK cells were earlier than that of GVHD group. Moreover,compared to the control groups, the plasma IL-2, IL-6, IFN-γ and TNF-α levels inrecipients infused with CXCR4over-expressed MSCs was significantly decreased,while those of IL-4and IL-10were increased.Conclusion: The lentiviral vector expressing CXCR4was successfully constructed.The lentiviral vector can not only infect mouse MSCs efficiently, but also couldstably express CXCR4in MSCs. The MSCs modified with CXCR4have biologicalcharacteristic in vitro. In vivo, MSCs can home to the spleen and bone marrow viatail vein injection. Moreover, infusion of CXCR4-MSC enhances the implantation ofhematopoietic stem cells and promotes hematopoietic recovery and immune recoveryafter syngeneic or allogeneic BMT. Furthermore, CXCR4-overexpressed MSCseffectively controlled the occurrence of mouse GVHD after allogeneic BMtransplantation... |
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