腺病毒介导hTRX修饰的间充质干细胞在小鼠急性放射损伤中的修复作用研究
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摘要
急性放射损伤的防治一直是各国重点开展研究的领域。目前造血干细胞移植和造血生长因子的应用仍是治疗极重度骨髓型急性放射病的主要措施，但作用有限。因此，急需寻找新的预防或治疗方法。硫氧还蛋白（TRX）具有清除自由基、抑制凋亡和促细胞生长等多重生物学作用，而间充质干细胞（MSC）易于外源基因转染和表达，且具有向照射后受损组织聚集的生物学特性。为此我们观察了腺病毒介导hTRX修饰的人脐带间充质干细胞（hucMSC）在急性放射损伤的NOD/SCID小鼠中的修复作用。为基因修饰的MSC治疗急性辐射相关性损伤提供了实验依据。经过相关文献的检索，目前尚未见国内外有相关研究的报道。
第一，我们采用骨片消化结合细胞贴壁法培养小鼠骨实质MSC；采用胶原酶消化法培养人脐带间充质干细胞（hucMSC），显微镜下观察细胞形态，细胞计数法绘制细胞生长曲线，采用流式细胞仪检测细胞周期及细胞表型，采用油红染色鉴定成脂诱导情况；阿尔辛蓝及甲苯胺蓝染色检测成软骨细胞诱导情况；碱性磷酸酶染色及VonKossa染色检测成骨细胞诱导情况。结果表明，分离、扩增培养的MSC细胞形态均一，其中小鼠骨实质MSC高表达Sca-1、CD29和CD44，而不表达CD11b、CD117、CD34和CD45；hucMSC细胞高表达HLA-I、CD105、CD166、CD73及CD90，不表达CD31、CD14、CD34、CD45、CD80、CD86及HLA-DR，符合MSC的表型特点。细胞周期检测显示，90%以上的细胞处于G0/G1期。在特定诱导条件下，两种MSC细胞均可向成骨、成软骨及脂肪细胞分化。
第二，我们以hTRX为目的基因，设计含有Not I和EcoR V酶切位点的引物，PCR扩增hTRX基因全长序列，将扩增产物连接到带有增强型绿色荧光蛋白(EGFP)标记的pDC316-mCMV穿梭质粒上，构建重组穿梭质粒pDC316-hTRX-EGFP，同时选用不含目的基因的空腺病毒质粒pAd316-EGFP作为对照。利用Lipofectamine2000脂质体转染的方法将AdMax腺病毒包装系统的骨架质粒pBHG lox_E1，3Cre和穿梭质粒pDC316-hTRX-EGFP共转染入HEK293细胞，进行同源重组，得到腺病毒重组质粒pAd-hTRX-EGFP，并在其中包装扩增病毒，将获得的病毒用氯化铯高速梯度离心、纯化病毒，测定病毒颗粒数及滴度。采用PCR方法对重组腺病毒进行鉴定。结果显示，重组腺病毒质粒经PCR和Not I、EcoR V酶切鉴定，证实含有hTRX基因，测序结果和TRX的理论序列一致。成功制备了纯度较高的病毒液，其中Ad-hTRX-EGFP病毒滴度达5.558×10~(10)pfu/ml，对照病毒Ad-EGFP病毒滴度达3.1×10~(10)pfu/ml。
第三，根据不同梯度的感染复数（MOI），我们检测了重组腺病毒载体Ad-EGFP对小鼠骨实质MSC的感染效率以及重组腺病毒载体Ad-hTRX-EGFP对293细胞和hucMSC的感染效率，用RealTime PCR检测了细胞内hTRX基因mRNA表达，采用Western blot检测了细胞内hTRX蛋白的表达。并探讨了感染hTRX基因对hucMSC细胞周期及分化特性的影响。结果显示，随着MOI值的升高，感染效率逐渐提高，当MOI=250时，Ad-EGFP对小鼠MSC的感染效率为46.78%；而当MOI=100时，Ad-hTRX-EGFP对293细胞和hucMSC的感染效率分别为92.5%和95.22%。Westernblot检测显示，感染后的293细胞和hucMSC细胞内hTRX蛋白量增高，hucMSC细胞内hTRX基因mRNA水平升高了10.52±3.21倍（P<0.05）。病毒感染后的hucMSC细胞周期显示，93.2%的细胞处于G0/G1期。在特定诱导条件下，感染后的hucMSC细胞可向软骨及脂肪细胞分化。说明感染hTRX对hucMSC的细胞周期及分化没有影响。
最后，我们评价了hTRX修饰的人脐带间充质干细胞在NOD/SCID小鼠急性放射损伤中的修复作用。将hTRX修饰的hucMSC通过尾静脉输注给经4.5Gy全身照射的NOD/SCID小鼠体内，并设置hucMSC治疗组和单纯照射组。通过观察小鼠一般状况和生存时间，检测血常规、骨髓Lin~-CD117~+细胞比例和组织病理来评价治疗效果。结果显示，照射后第7天、第11天、第20天及第30天hTRX-hucMSC组小鼠的红细胞及血红蛋白显著高于单纯照射组和hucMSC组小鼠，其中第11天及第30天，hTRX-hucMSC组与其他两组均存在统计学差异（P＜0.05）；而第7天及第20天仅hTRX-hucMSC组与单纯照射组存在统计学差异（P＜0.05）。虽然第7天、第20天及第30天hucMSC组小鼠的红细胞及血红蛋白也高于单纯照射组，但二者之间无统计学差异。+30天hTRX-hucMSC组小鼠骨髓Lin-CD117+细胞比例明显高于其他两组，且均存在统计学差异（P＜0.05）。各组小鼠照射30天组织病理显示，肝、脾和肠病理改变相对较轻微，而骨髓及肺脏病理改变显著。单纯照射组肝细胞轻度水肿，肝动脉及门静脉充血，可见胆栓形成；脾脏可见血管充血及大量炎细胞浸润。hucMSC组和hTRX-hucMSC组肝脾病理表现较单纯照射组略减轻。肠粘膜病理各组无明显差异。单纯照射组小鼠肺泡壁显著增宽，血管显著充血，出血，间质水肿，透明膜形成，炎细胞浸润显著。hucMSC组小鼠肺泡壁增宽，血管充血，中等量炎细胞浸润。而hTRX-hucMSC组小鼠肺泡壁间隔正常，少许血管充血，少量炎细胞浸润。提示hTRX-hucMSC组小鼠肺损伤比其他两组小鼠明显减轻。单纯照射组小鼠骨髓组织增生极度减低；hucMSC组小鼠骨髓组织增生轻度减低；而hTRX-hucMSC组小鼠骨髓增生活跃。各组生存时间显示，单纯照射组小鼠生存时间为26±14.43d；hucMSC组为29.33±14.49d，生存时间较空白组延长，但无统计学差异（P=0.36），而hTRX-hucMSC组小鼠为56.67±5.25d。生存时间较前两组显著延长（P<0.01）。以上结果说明，hTRX通过保护了残存的造血干细胞，特别是促进红细胞及血红蛋白生成，来保证重要组织脏器的氧供应。此外，通过清除自由基及调节炎症反应，减轻肺损伤。因此能够延长小鼠生存时间。
以上研究结果表明，腺病毒能够高效的感染hucMSC；腺病毒介导hTRX基因能在hucMSC中高表达；hTRX基因修饰的hucMSC能够减轻急性放射损伤引起的组织损伤，对急性放射损伤的治疗具有较好的效果。本研究将基因治疗与细胞治疗相结合，为治疗急性放射损伤提供了新的可能手段。
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 was5.558×10~(10)pfu/ml and Ad-EGFP control was3.1×10~(10)pfu/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.
第一，我们采用骨片消化结合细胞贴壁法培养小鼠骨实质MSC；采用胶原酶消化法培养人脐带间充质干细胞（hucMSC），显微镜下观察细胞形态，细胞计数法绘制细胞生长曲线，采用流式细胞仪检测细胞周期及细胞表型，采用油红染色鉴定成脂诱导情况；阿尔辛蓝及甲苯胺蓝染色检测成软骨细胞诱导情况；碱性磷酸酶染色及VonKossa染色检测成骨细胞诱导情况。结果表明，分离、扩增培养的MSC细胞形态均一，其中小鼠骨实质MSC高表达Sca-1、CD29和CD44，而不表达CD11b、CD117、CD34和CD45；hucMSC细胞高表达HLA-I、CD105、CD166、CD73及CD90，不表达CD31、CD14、CD34、CD45、CD80、CD86及HLA-DR，符合MSC的表型特点。细胞周期检测显示，90%以上的细胞处于G0/G1期。在特定诱导条件下，两种MSC细胞均可向成骨、成软骨及脂肪细胞分化。
第二，我们以hTRX为目的基因，设计含有Not I和EcoR V酶切位点的引物，PCR扩增hTRX基因全长序列，将扩增产物连接到带有增强型绿色荧光蛋白(EGFP)标记的pDC316-mCMV穿梭质粒上，构建重组穿梭质粒pDC316-hTRX-EGFP，同时选用不含目的基因的空腺病毒质粒pAd316-EGFP作为对照。利用Lipofectamine2000脂质体转染的方法将AdMax腺病毒包装系统的骨架质粒pBHG lox_E1，3Cre和穿梭质粒pDC316-hTRX-EGFP共转染入HEK293细胞，进行同源重组，得到腺病毒重组质粒pAd-hTRX-EGFP，并在其中包装扩增病毒，将获得的病毒用氯化铯高速梯度离心、纯化病毒，测定病毒颗粒数及滴度。采用PCR方法对重组腺病毒进行鉴定。结果显示，重组腺病毒质粒经PCR和Not I、EcoR V酶切鉴定，证实含有hTRX基因，测序结果和TRX的理论序列一致。成功制备了纯度较高的病毒液，其中Ad-hTRX-EGFP病毒滴度达5.558×10~(10)pfu/ml，对照病毒Ad-EGFP病毒滴度达3.1×10~(10)pfu/ml。
第三，根据不同梯度的感染复数（MOI），我们检测了重组腺病毒载体Ad-EGFP对小鼠骨实质MSC的感染效率以及重组腺病毒载体Ad-hTRX-EGFP对293细胞和hucMSC的感染效率，用RealTime PCR检测了细胞内hTRX基因mRNA表达，采用Western blot检测了细胞内hTRX蛋白的表达。并探讨了感染hTRX基因对hucMSC细胞周期及分化特性的影响。结果显示，随着MOI值的升高，感染效率逐渐提高，当MOI=250时，Ad-EGFP对小鼠MSC的感染效率为46.78%；而当MOI=100时，Ad-hTRX-EGFP对293细胞和hucMSC的感染效率分别为92.5%和95.22%。Westernblot检测显示，感染后的293细胞和hucMSC细胞内hTRX蛋白量增高，hucMSC细胞内hTRX基因mRNA水平升高了10.52±3.21倍（P<0.05）。病毒感染后的hucMSC细胞周期显示，93.2%的细胞处于G0/G1期。在特定诱导条件下，感染后的hucMSC细胞可向软骨及脂肪细胞分化。说明感染hTRX对hucMSC的细胞周期及分化没有影响。
最后，我们评价了hTRX修饰的人脐带间充质干细胞在NOD/SCID小鼠急性放射损伤中的修复作用。将hTRX修饰的hucMSC通过尾静脉输注给经4.5Gy全身照射的NOD/SCID小鼠体内，并设置hucMSC治疗组和单纯照射组。通过观察小鼠一般状况和生存时间，检测血常规、骨髓Lin~-CD117~+细胞比例和组织病理来评价治疗效果。结果显示，照射后第7天、第11天、第20天及第30天hTRX-hucMSC组小鼠的红细胞及血红蛋白显著高于单纯照射组和hucMSC组小鼠，其中第11天及第30天，hTRX-hucMSC组与其他两组均存在统计学差异（P＜0.05）；而第7天及第20天仅hTRX-hucMSC组与单纯照射组存在统计学差异（P＜0.05）。虽然第7天、第20天及第30天hucMSC组小鼠的红细胞及血红蛋白也高于单纯照射组，但二者之间无统计学差异。+30天hTRX-hucMSC组小鼠骨髓Lin-CD117+细胞比例明显高于其他两组，且均存在统计学差异（P＜0.05）。各组小鼠照射30天组织病理显示，肝、脾和肠病理改变相对较轻微，而骨髓及肺脏病理改变显著。单纯照射组肝细胞轻度水肿，肝动脉及门静脉充血，可见胆栓形成；脾脏可见血管充血及大量炎细胞浸润。hucMSC组和hTRX-hucMSC组肝脾病理表现较单纯照射组略减轻。肠粘膜病理各组无明显差异。单纯照射组小鼠肺泡壁显著增宽，血管显著充血，出血，间质水肿，透明膜形成，炎细胞浸润显著。hucMSC组小鼠肺泡壁增宽，血管充血，中等量炎细胞浸润。而hTRX-hucMSC组小鼠肺泡壁间隔正常，少许血管充血，少量炎细胞浸润。提示hTRX-hucMSC组小鼠肺损伤比其他两组小鼠明显减轻。单纯照射组小鼠骨髓组织增生极度减低；hucMSC组小鼠骨髓组织增生轻度减低；而hTRX-hucMSC组小鼠骨髓增生活跃。各组生存时间显示，单纯照射组小鼠生存时间为26±14.43d；hucMSC组为29.33±14.49d，生存时间较空白组延长，但无统计学差异（P=0.36），而hTRX-hucMSC组小鼠为56.67±5.25d。生存时间较前两组显著延长（P<0.01）。以上结果说明，hTRX通过保护了残存的造血干细胞，特别是促进红细胞及血红蛋白生成，来保证重要组织脏器的氧供应。此外，通过清除自由基及调节炎症反应，减轻肺损伤。因此能够延长小鼠生存时间。
以上研究结果表明，腺病毒能够高效的感染hucMSC；腺病毒介导hTRX基因能在hucMSC中高表达；hTRX基因修饰的hucMSC能够减轻急性放射损伤引起的组织损伤，对急性放射损伤的治疗具有较好的效果。本研究将基因治疗与细胞治疗相结合，为治疗急性放射损伤提供了新的可能手段。
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 was5.558×10~(10)pfu/ml and Ad-EGFP control was3.1×10~(10)pfu/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.
引文
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