移植物化学修饰减轻小鼠半相合骨髓移植后的急性移植物抗宿主病

背景:甲氧基聚乙二醇是一种无免疫原性的兼性聚合分子,可以共价结合方式修饰各种蛋白质,减少其免疫原性.因此,在进行半相合造血干细胞移植时,若能用甲氧基聚乙二醇对移植物的T细胞进行化学修饰,阻断其激活的途径,就可能减轻移植物抗宿主病反应,提高移植成功率.目的:构建小鼠半相合骨髓移植模型,观察甲氧基聚乙二醇修饰移植物对小鼠H2半相合骨髓移植后移植物抗宿主病的影响.设计、时间及地点:随机化配对设计实验,于2003-03/11在解放军总医院医学动物实验中心和小儿内科实验室完成.材料:纳入20只8～10周龄雄性近交系BALB/cH-2d小鼠作为供鼠,60只8-10周龄杂交获得的雌性CB6 F1代".m小鼠作为受鼠.甲氧基聚乙二醇为美国Sigma公司产品.方法:常规制备供鼠骨髓及脾细胞混合悬液.受鼠行总剂量8.0 GY60Co γ射线全身均匀照射,照射后随机分为3组,每组20只.照射对照组每只受鼠经尾静脉输入RPMI-1640培养摹基0.5 mL,单纯移植组照射后12 h内经尾静脉输入未处理的半相合供鼠骨髓及脾细胞混合悬液0.5 mL,修饰移植组照射后12 h内经尾静脉输入甲氧基聚乙二醇修饰的半相合供鼠骨髓及脾细胞混合悬液0.5mL.主要观察指标:移植后观察动物存活率、急性移植物抗宿主病理表现及存活小鼠的染色体核型.结果:照射对照组小鼠均在2周内死亡;修饰移植组小鼠30 d存活率显著高于单纯移植组(75%,40%,x2=5.01,P=0.025).取死亡小鼠的爪垫皮肤、肝及小肠肠管作病理组织学检查,修饰移植组急性移植物抗宿主病病理表现减轻,Thomas病理分级轻于单纯移植组.移植后75 d应片j染色体C带显色法检测存活受鼠的嵌合体,证实为完全供者型植入.结论:甲氧基聚乙二醇修饰移植物可明显减轻急性移植物抗宿主病,提高半相合骨髓移植存活率.

Modification with methoxy polyethylene glycol to grafts alleviates acute graft versus host disease severity in mice following haploidentical bone marrow transplantation

BACKGROUND: Methoxy polyethylene glycol (mPEG) is a kind of amphotedc compound with no immunogenicity that has been used to modify various proteins covalently and to prepare versatile blood types. If mPEG modification blocks the activation of T cells in grafts, graft versus host disease (GVHD) reaction probably becomes less serious and transplantation may become successful. OBJECTIVE: To construct haploidentical bone marrow transplantation mudne model and to observe the effects of mPEG modification to grafts on acute GVHD following haploidentical bone marrow transplantation, DESIGN, TIME AND SETTING: A randomized paired design experiment was performed at the Laboratory Animal Center and Pediatric Laboratory of the General Hospital of Chinese PLA between March and November 2003. MATERIALS: Twenty 8-10 week old male BALB/cH-2d mice served as donors and sixty 8-10 week old female CBe F1H-2d/b mice served as recipients, mPEG was provided by Sigma, USA. METHODS: Mixture of donor bone marrow and spleen cells was routinely prepared. After irradiated with 60Co γ ray at a total dose of 8.0 Gy, recipient mice were randomly divided into 3 groups, with 20 rats per group: irradiation control, non-modification, and mPEG modification. Within 12 hours after irradiation, the irradiation control group was injected with 0.5 mL RPMI-1640 culture medium via caudal vein, the non-modification group was administered with 0.5 mL non-modified mixture of bone marrow and spleen cells via caudal vein, and mice from the group were given 0.5 mL mPEG-modified mixture of bone marrow and spleen cells via caudal vein. MAIN OUTCOME MEASURES: After transplantation, hematopoietic recovery, survival rate, acute GVHD and chromosomal karyotype were studied and compared with controls. RESULTS: All mice from the irradiation control groups died within 2 weeks. The 30-day survival rate was significantly higher in the mPEG modification group than in the non-modffication group (75% vs. 40%, x2 = 5.01, P= 0.025). Histopathological examinations of skin, liver and intestine showed typical signs of acute GVHD. The mPEG modification group exhibited less severe pathological presentation and lower Thomas grading than the non-modification group. Cheimedsm examination revealed complete donor-type implantation in living recipient mice at 75 days after transplantation. CONCLUSION: mPEG modification to grafts can greatly alleviate acute GVHD and enhance the survival rate of mice after haploidentical bone marrow transplantation.