Treatment Effect Of Mice With Acute Radiation Injury By HTRX-modified Mesenchymal Stem Cells

Prevention and treatment of acute radiation injury has been the focus of medicalresearches in many countries. Hematopoietic stem cell transplantation andhematopoietic growth factors are still the main measures to treat extremely severe acuteradiation diseases, but has limited effects. Thus,it is crucial to find a alternative methodto improve the treatment. Thioredoxin (TRX) can scavenge free radical and inhibitapoptosis and promote cell growth. Mesenchymal stem cells (MSCs) can easily beinfected by exogenous gene and home to radiated tissue. In this study, we observed thetreatment of hTRX-modified human umbilical cord mesenchymal stem cells (hucMSCs)in NOD/SCID mice with acute radiation injury. This study will lay a foundation fortreating acute radiation injury by genetically-modified MSCs.Firstly, murine MSCs were isolated from compact bone, cultured and expanded invitro by using bone slices digestion combined with cell adhesion. Human umbilical cordmesenchymal stem cells (hucMSCs) were isolated by using a collagenase digestionmethod and expanded in vitro. Cell morphology was observed by the microscope, thegrowth curve was drawn by cell counting, and the cell cycle and the phenotypes wereevaluated by flow cytometry. Oil red staining was used to determine the differentiationinto adipocytes,and alcian blue staining and toluidine blue staining were used todetermine the differentiation into chondrocytes. Alkaline phosphatase staining and theVonKossa staining were used to determine the differentiation into osteoblasts. Theresults showed that isolated and expanded MSCs had the properties of plastic adherenceand homogeneity. Murine MSCs displayed an abundant presence of Sca-1, CD29,CD44and absence of CD11b, CD117, CD34andCD45, while hucMSCs displayed anabundant presence of HLA-I, CD105, CD166, CD73, CD90and absence of CD31,CD14, CD34, CD45, CD80, CD86and HLA-DR. It has shown that more than90%ofcells were in the G0/G1phase. Murine MSCs and hucMSCs have the ability todifferentiate into osteoblasts, chondrocytes and adipocytes in vitro.Secondly, as a target gene, the amplification product of hTRX by PCR with a pairof primers with Not I and EcoR V restriction endonuclease sites was subcloned into shuttle plasmid pDC316-mCMV marked with EGFP.293cells were co-transfected withthe constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and largeadenovirus helper plasmid pBHGlox(delta)E1,3Cre in mediation of liposome. Theobtained recombinant adenovirus pAd-hTRX-EGFP were propagated in293cells,purified by CsCl gradient centrifugation, and counted for virus particles and determinedfor titer. The recombinant adenovirus were identified by PCR. The result of PCR andrestriction endonuclease Not I and EcoR V assay indicated that target gene was insertedinto recombinant adenovirus vector successfully. The sequence of fusion gene was thesame as that of designed fragments. The titer of the purified recombinant adenovirusAd-hTRX-EGFP was5.558×10¹⁰pfu/ml and Ad-EGFP control was3.1×10¹⁰pfu/ml.Thirdly, according to the multiplicity of infection (MOI), the infection efficiencyof the murine MSC with Ad-EGFP, the293cells and hucMSCs with Ad-hTRX-EGFPwere detected by flow cytometry. Expression level of hTRX gene mRNA was analysedby Realtime PCR. The hTRX protein was measured by Western blot. Cell cycle,immune phenotype and differentiation characteristics of the hTRX-modified hucMSCswere alse detected. The results showed that the infection efficiency increased graduallyby the rise of MOI values. As MOI=250, the infection efficiency of the murine MSCsinfected by Ad-EGFP was46.78%. As MOI=100, the infection efficiency of the293cells and hucMSCs infected by Ad-hTRX-EGFP was92.5%and95.22%respectively.Western blot analysis showed that hTRX protein expressed highly in infected293cellsand hucMSCs. The mRNA expression of hTRX gene increased to10.52±3.21times(p<0.05) in infected hucMSCs. It has shown that more than93.21%of cells werein the G0/G1phase. The infected hucMSCs have the ability to differentiate intoosteoblasts, chondrocytes and adipocytes in vitro. hTRX did not affect on theproliferation and differentiation characteristics of hucMSCs.Finally, it was evaluated that the treatment effect of the hTRX-modified humanumbilical cord mesenchymal stem cells in NOD/SCID mice with acute radiation injury.The hTRX-modified hucMSCs were infused by tail vein to the NOD/SCID mice with4.5Gy irradiation, and mice was randomly divided into the irradiation group, hucMSCstreatment group and hTRX-modified hucMSCs group. The general condition of mice,survival time, the detection of blood, bone marrow Lin-CD117+cell ratio andhistopathology were evaluated as the therapeutic effect. The results showed that thenumber of the red blood cells and the level of hemoglobin of hTRX-hucMSCs treatment mice were significantly higher than that of the irradiation group and that of thehucMSCs mice on the7^th day,11^th day,20^th day and30^th day after irradiation. Therewere statistically significant differences between the hTRX-hucMSCs group and theother two groups (P <0.05) on the11^th day and30^th day, while similar results were onlyfound between the hTRX-hucMSCs group and the irradiation group (P <0.05) on the7^th day and20^th day, but no statistically significant differences between the hucMSCsgroup and the irradiation group on the7^th day,20^th day and30^th day. On the30^th Day,the Lin^-CD117⁺cells of bone marrow in hTRX-hucMSC group were significantlyhigher than that of the other two groups(P <0.05). On the30^th days histopathologicalchanges were slight in the liver, spleen and intestinal, while significant in the bonemarrow and lungs. There were mild edema of the liver cells, hepatic artery and portalvein congestion and biliary embolism in the irradiation group. The vascular congestionand inflammatory cell infiltration were observed in spleen. The histopathologicalchanges of the other two groups were slighter than the irradiation group in the liver andspleen. Intestinal mucosa did not show obvious histopathologically change in eachgroup. The widened significantly alveolar wall, vascular congestion, hemorrhage,interstitial edema, hyaline membrane formation and inflammatory cell infiltration weresignificantly observed in the mice of irradiation group. The interval of mice alveolarwall was normal, and vascular congestion and infiltration of inflammatory cells wasslight in the hTRX-modified group. The changes of lung in the hucMSC group werebetween the other two groups. It suggested that lung injury in the hTRX-modified groupsignificantly decreased than the other groups. The proliferation of mouse bone marrowtissue was extremely decreased in the irradiation group, and was slightly decreased inhucMSCs group. The bone marrow of hTRX-modified group proliferated actively. Thesurvival time of the hucMSCs group was29.33±14.49d longer than that of theirradiation group26±14.43d, but no statistics difference (p=0.36). The survival time ofhTRX-modified group was56.67±5.25d longer than that of the other groups (p<0.01). Itwas suggested that hTRX supplied oxygen for the main organs and tissues by protectingthe remnants of hematopoietic stem cells, especially the formation of red blood cellsand hemoglobin. Furthermore, hTRX reduced lung injury by scavenging free radicalsand regulating the inflammatory response and therefore increased survival time.To summarize, our data signified that human umbilical cord mesenchymal stemcells could easily be infected by adenovirus carrying hTRX gene. A high level of expression of hTRX was achieved by hTRX gene modified hucMSCs. hTRX-modifiedhucMSCs could alleviate tissue injury caused by acute radiation injury. Thus, therapy ofhTRX gene combined with hucMSCs may be provide a new choice for the treatment ofacute radiation injury.