CAJ | 학술논문

目的探讨联合输注经Wnt3a基因修饰的MSC减轻小鼠异基因骨髓移植(allo-BMT)后急性移植物抗宿主病(aGVHD)的作用及其可能机制.方法以C57BL/6小鼠为供鼠,以Balb/c小鼠为受鼠,建立小鼠allo BMT模型.采用随机数字表法将受鼠分为4组:(1)移植对照组(A组):经受鼠尾静脉仅输注供鼠骨髓细胞5×106个；(2) aGVHD组(B组):经受鼠尾静脉输注供鼠脾细胞5×106个及骨髓细胞5×106个；(3)aGVHD+空载体组(C组):经受鼠尾静脉输注供鼠脾细胞5×106个、骨髓细胞5×106个及转染了空载体pAd-GFP的MSC 1×106个；(4)实验组(D组):经受鼠尾静脉输注供鼠脾细胞5×106个、骨髓细胞5×106个及经Wnt3a基因修饰的MSC1×106个.移植后监测各组受鼠的一般表现和存活情况,观察aGVHD的发生情况,检测受鼠脾脏中供者来源的T淋巴细胞数量变化及白细胞介素2(IL-2)和γ干扰素(IFN-γ)水平.结果 A组受鼠的存活时间均超过60d；B、C、D组受鼠的存活时间分别为(19.1±6.19)d、(32.6±19.6)d和(47.2±15.6)d,D组受鼠的存活时间较B组和C组明显延长(P＜0.05).移植后,B、C、D组受鼠的aGVHD评分分别为(8.0±0.41)分、(6.7±0.29)分和(4.0±1.0)分,D组受鼠的aGVHD评分较B组和C组明显降低(P＜0.05),且病理分级明显减轻.移植后3和5d时D组受鼠脾脏中供者T淋巴细胞的数量和增殖速度均较B组和C组明显降低(P＜0.05),并且移植后7、14、21、28 d时D组受鼠血清IL-2和IFN-γ水平均较B组和C组明显减少(P＜0.05).术后60d时,长期存活受鼠的骨髓细胞中H-2Kb细胞的嵌合率均在95％～100％.结论联合输注经Wnt3a基因修饰的MSC可更有效的减轻小鼠allo-BMT后的aGVHD,这可能与Wnt3a的过表达激活了MSC的Wnt/β-catenin信号通路,从而抑制供者T淋巴细胞的早期激活和扩增及抑制IL-2和IFN-γ的表达有关.
목적 탐토연합수주경Wnt3a기인수식적MSC감경소서이기인골수이식(allo-BMT)후급성이식물항숙주병(aGVHD)적작용급기가능궤제.방법 이C57BL/6소서위공서,이Balb/c소서위수서,건립소서allo BMT모형.채용수궤수자표법장수서분위4조:(1)이식대조조(A조):경수서미정맥부수주공서골수세포5×106개；(2) aGVHD조(B조):경수서미정맥수주공서비세포5×106개급골수세포5×106개；(3)aGVHD+공재체조(C조):경수서미정맥수주공서비세포5×106개、골수세포5×106개급전염료공재체pAd-GFP적MSC 1×106개；(4)실험조(D조):경수서미정맥수주공서비세포5×106개、골수세포5×106개급경Wnt3a기인수식적MSC1×106개.이식후감측각조수서적일반표현화존활정황,관찰aGVHD적발생정황,검측수서비장중공자래원적T림파세포수량변화급백세포개소2(IL-2)화γ간우소(IFN-γ)수평.결과 A조수서적존활시간균초과60d；B、C、D조수서적존활시간분별위(19.1±6.19)d、(32.6±19.6)d화(47.2±15.6)d,D조수서적존활시간교B조화C조명현연장(P＜0.05).이식후,B、C、D조수서적aGVHD평분분별위(8.0±0.41)분、(6.7±0.29)분화(4.0±1.0)분,D조수서적aGVHD평분교B조화C조명현강저(P＜0.05),차병리분급명현감경.이식후3화5d시D조수서비장중공자T림파세포적수량화증식속도균교B조화C조명현강저(P＜0.05),병차이식후7、14、21、28 d시D조수서혈청IL-2화IFN-γ수평균교B조화C조명현감소(P＜0.05).술후60d시,장기존활수서적골수세포중H-2Kb세포적감합솔균재95％～100％.결론 연합수주경Wnt3a기인수식적MSC가경유효적감경소서allo-BMT후적aGVHD,저가능여Wnt3a적과표체격활료MSC적Wnt/β-catenin신호통로,종이억제공자T림파세포적조기격활화확증급억제IL-2화IFN-γ적표체유관.
Objective To explore the effects of injection of wnt3a gene-modified bone marrow mesenchymal stem cells (MSCs) on acute graft-versus-host disease (aGVHD) in a murine allogeneic bone marrow transplantation (allo-BMT) model.Methods C57BL/6 mice were used as the donors and Balb/c mice as the recipients in the murine allo-BMT model.The recipient mice were divided into four groups by random number table method: transplantation control group (group A) (infusion of 5 × 106bone marrow cells via the tail vein of recipient mice); aGVHD group (group B) (infusion of 5 × 106bone marrow cells and 5 × 106 splenocytes via the tail vein of recipient mice); aGVHD + empty vector group (group C) (infusion of 5 × 106 bone marrow cells,5 × 106 splenocytes and 1 × 106 pAd-GFP-transfected MSCs via the tail vein of recipient mice) ; experimental group (group D) (infusion of 5 ×106 bone marrow cells,5 × 106 splenocytes and 1 × 106 wnt3a gene-modified MSCs).The general performance and survival were monitored,the occurrence of aGVHD was observed,the changes of donor T lymphocyte quantity present in the spleen,and interleukin-2 (IL-2) and interferon-γ (IFN γ)levels of the recipient mice were detected in each group after transplantation.Results The survival time of recipient mice in group A was all more than 60 d,and that in groups B,C and D was (19.1 ±6.19),(32.6 ± 19.6) and (47.2 ± 15.6) d,rcspcctivcly.The survival time in group D was significantly longer than in groups B and C (P＜0.05).After the transplant,the aGVHD score points in groups B,CandDwere (8.0±0.41),(6.7±0.29) and (4.0± 1.0),respcctively.The aGVHD score points in group D were significantly less than in groups B and C (P＜0.05),and the pathological grade in group D was significantly reduced.The number and proliferation rate of T lymphocytes were reduced significantly in group D as compared with groups B and C at 3rd and 5th day after transplantation (P ＜ 0.05).The levels of IL-2 and IFN-γ in peripheral blood were decreased significantly in group D as compared with those in groups B and C at 7th,14th,21st and 28th day after transplantation (P＜0.05).The chimeric rate of the murine H-2Kb cells in the bone marrow cells of long-term survival mice was all in the range of 95％ to 100％ 60 d after transplantation.Conclusion The injection of wnt3a gene-modified MSCs can more effectively alleviate aGVHD in murineallo-BMT model,which may be correlated with the Wnt3a overexpression which activating the Wnt/β-catenin signaling pathway of MSCs,thereby inhibiting the early activation and amplification of donor T lymphocytes and the IL-2 and IFN-γ expression.