大鼠同种异体骨髓移植慢性移植物抗宿主疾病模型的建立

Establishment of chronic graft-versus-host disease models after allo-bone marrow transplantation in rats

目的:探讨同种大鼠骨髓移植建立慢性移植物抗宿主疾病模型的可行性,旨在为骨髓间质干细胞对同种异体骨髓移植后移植物抗宿主疾病免疫调节作用的系列研究提供前提条件。方法:实验于2006-04/2006-09在中山大学基础医学院病理学与病理生理实验室完成。①选取清洁级6周龄雄性体Fischer344大鼠(RT1Al)作为供体,清洁级6周龄雌性Waster(RT1Au)大鼠作为受体。②受体大鼠移植前3d开始饮用含庆大霉素(320mg/L)和红霉素(250mg/L)的饮用水。移植当天采用60Coγ射线全身照射法进行预处理,照射剂量为8.0Gy,剂量率为0.7Gy/min。经预处理的受体鼠在照射后6h内,经尾静脉进行骨髓移植,并分组:骨髓移植组(n=10,每只大鼠移植0.8×108个骨髓细胞)、单纯照射组(n=10,预处理后通过尾静脉注射等量的磷酸缓冲液)。观察移植后大鼠的活动、饮食、二便、皮毛、体质量变化情况及靶器官皮肤、肝脏、肠的组织学病理改变。结果:①单纯照射组10只大鼠在预处理后的17d内全部死亡,死亡高峰集中在移植后11和12d左右。骨髓移植组有较高的存活率,除第12,16,18天死亡3只,其余均在60d的观察期内存活。②骨髓移植组大鼠未出现急剧消瘦和严重腹泻等急性移植物抗宿主疾病表现,移植受体体质量增加缓慢,移植后1月受体头背部可见明显脱毛现象,随着时间延长,范围逐渐扩大至腹部和肢体内侧。移植后2个月皮肤可见毛囊缺失和轻度单核细胞浸润,小肠固有层可有中性粒细胞、嗜酸性粒细胞、巨噬细胞等炎症细胞浸润,肝脏的表现最为典型,80%以上的汇管区淋巴细胞浸润,胆管细胞坏死并伴有胆管壁纤维化。结论:①通过大鼠F344→Wistar的同种骨髓移植,可建立稳定的慢性移植物抗宿主疾病模型。②模型的病理变化可表现在皮肤、肠道和肝脏,其中肝脏的表现尤其典型,80%以上的汇管区和胆管淋巴细胞浸润,胆管细胞坏死并伴有胆管壁纤维化。

AIM： To investigate the feasibility of establishment for chronic graft-versus-host disease （cGVHD） model in rats after allo-bone marrow transplantation （allo-BMT）, in order to provide premise conditions for further studying the immuno-regulation role of mesenchymal stem cell （MSC） on GVHD after alIo-BMT. METHODS： This experiment was finished in Laboratory of Pathology and Pathophysiology in Sun Yat-sen University from April to September 2006. （1）Six-week-old male Fischer344 rats （RTIAI） were used as donors while six-week-old female Waster （RTIAu） rats were used as recipients.（2）Recipient rats were given water supplemented with gentamycin （320 mg/L） and erythromycin （250 mg/L） three days before BMT. On the day of transplantation, recipient rats received 8.0 Gy （^60Co γ, 0.7 Gy/min） total body irradiation. Within 6 hours following the irradiation, recipient rats in BMT group were transplanted with 0.8×10^8 cells via tail vein injection, while rats in control group only received the same volume injection of phosphate buffer. Each group included 10 animals. Evaluation of common living status was monitored including daily diet, activity, stool and urine, fur and body mass. Shaved skin, liver and intestine tissues were also analyzed histologically. RESULTS：（1）AII rats in the control group died within 17 days following the irradiation and most of them died on day 11 or 12 post-transplantation, while BMT group had higher survival rate, in addition to three rats died on days 12, 16, 18 respectively, whereas others were all alive through 60 days expectation period. （2）Rats in the BMT group had no clear symptoms of acute GVHD, such as rapid weight loss and severe diarrhea, however, the weight growth in rats of the BMT group was quite slow. Furthermore, 1 month following BMT, depilation phenomenon was evident in the head and back of recipients, and then extended to abdominal part and extremity with the increase of time. Two months following BMT, skin follicular dropout and slight dermal mononuclear infiltration were found. Hepatic disease was characterized by portal tract lymphocyte infiltration, fibrous thickening and sclerosis of the bile duct wall. Small bowel specimens showed clear inflammatory cell infiltration （neutrophils, acidophils, macrophages） within lamina propria. CONCLUSION： （1）The cGVHD model can be established through alIo-BMT from F344 to Wistar rats. （2）The typical histological signs of cGVHD are evident in skin, liver and intestine tissues, among which hepatic sign is the most dominant including portal tract and bile duct mononuclear infiltration followed by fibrous thickening and sclerosis of the bile duct wall.