利用脊柱骨来源骨髓细胞建立急性移植物抗宿主病模型

Establishment of the model of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation by backbone derived bone marrow cells

目的探讨利用脊柱骨来源骨髓细胞建立小鼠异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,Allo-HSCT)急性移植物抗宿主病(aGVHD)模型的可行性。方法选择C57BL/6(H-2b)雄性小鼠为供体鼠,BALB/c(H-2d)雌性小鼠为受体鼠。制备供体鼠的脾细胞和脊柱骨来源骨髓细胞悬液。受体鼠采用药物加小剂量辐照的预处理方式,于移植前8d～移植前4d腹腔注射氟达拉滨(200mg/kg),接着移植前3d～移植前1d腹腔注射环磷酰胺(60mg/kg),最后在移植前进行全身照射(total-body irradiation,TBI),照射剂量为4Gy(戈瑞)。18只受体鼠经预处理后随机分为3组,每组6只:(1)骨髓移植组,只输入1×107个脊柱骨来源骨髓细胞;(2)aGVHD组,输注1×107个脊柱骨来源骨髓细胞和5×106个脾细胞,建立aGVHD模型;(3)空白对照组,不输入任何细胞。观察3组小鼠生存状态及存活率,取aGVHD组与骨髓移植组存活21d的受体鼠进行病理学检查,取aGVHD组移植后21～28d存活的小鼠的脾脏进行流式细胞术检测骨髓细胞嵌合度。结果骨髓移植组小鼠全部存活,可重建造血,单纯输注骨髓细胞不会诱发aGVHD。aGVHD组小鼠出现aGVHD表现,100%发生aGVHD相关死亡,中位生存期为18d;病理检查结果显示符合aGVHD病理表现,移植后21～28d存活的小鼠诊断为供受体混合嵌合状态,符合aGVHD诊断标准。结论用脊柱骨来源骨髓建立的aGVHD模型完全符合标准,且更加经济,适合大规模建模。

Objective To investigate the feasibility in establishing the mice model of acute graft- versus-hest disease （aGVHD） after allogeneic hematopoietic stem cell transplantation （Allo-HSCT） by using backbone derived bone marrow cells. Methods C57BL/6 （ H-2^b ） male mice were chosen as donors and BALB/c （ H-2^d） female mice as recipients. The suspension of splenocytes and backbone derived bone marrow cells from donor mice were prepared. Drug and low dose of irradiation were used as pretreatment. Fludarabine （200 mg/kg） and cyclophosphamide （60 mg/kg） were injected intraperitoneally from preoperative 8 to 4 days and preoperative 3 to 1 days respectively. Then 4 Gy of total-body irradiation （TBI） was performed before transplantation. Eighteen recipient mice were randomly divided into three groups after pretreatment by 6 in each group： （1） bone marrow transplantation （BMT） group, 10×10^7 bone marrow cells （BMC） were injected; （2） aGVHD group, 10 ×10^7 BMC and 5 × 10^6 splenocytes were injected; （3） control group, no ceils were injected. The postoperative survival rate and outcome were observed in all mice. The pathologic examination was performed on the recipient mice that survived 21 days in aGVHD and BMT group. The chimeric degree of donor BMC in spleen of recipient mice that survived 21-28 days after transplantation in aGVHD group wasdetected by flow cytometry. Results In BMT group, all mice survived and their hematopoiesis were reestab- lished. BMC infusion alone wouldn＇t lead to aGVHD. In aGVHD group, ＇all mice developed the aGVHD symp- toms and died of aGVHD. The median survival time was 18 days. The pathologic results showed typical aGVHD performance. The chimerism status was detected in these mice that survived 21-28 days after transplantation and confirmed the diagnosis of aGVHD. Conclusions The aGVHD model established by using backbone derived bone marrow cells is up to standard, economic and suitable for large-scale production. Conclusions The aGVHD model established by using backbone derived bone marrow cells is up to standard, economic and suitable for large-scale production.