摘要
目的：因供受者主要或次要组织相容性抗原存在差异而引起的移植物抗宿主病是影响异基因造血干细胞移植效果的关键因素,目前国内尚缺乏研究此领域的实验动物模型及其评价体系。本实验拟建立半相合异基因造血干细胞移植后慢性移植物抗宿主病小鼠模型,并制定半定量评价体系。方法：实验于2007-03/08在南方医科大学实验动物中心完成。①动物：供鼠为近交系Balb/CH-2d小鼠40只,雄性,8～10周龄,体质量18～22g;受鼠为CB6F1小鼠（Balb/c×C57BL/6F1H-2d/b）48只,雌性,10～12周龄,体质量18～24g,随机数字表法分为3×10^7,6×10^7,9×10^7个脾细胞移植组及空白对照组,12只/组。小鼠均由南方医科大学实验动物中心提供,SPF级,实验过程中对动物的处置符合动物伦理学标准。②实验方法：Balb/CH-2d小鼠颈椎脱臼处死,取脾脏研磨后制成脾单细胞悬液,离心弃上清,加入Tris-NH4Cl裂解红细胞,用RPMI1640调整细胞浓度为1.2×10^8L^-1,锥虫蓝染色计数脾细胞拒染率为95.6%。3×10^7,6×10^7,9×10^7个脾细胞移植组分别经尾静脉输入对应数量的MHC半相合脾细胞悬液0.5mL,空白对照组输入等体积RPMI1640培养液。③实验评估：脾细胞移植后2,5,8,12周,光镜下计数20个分裂相,检查骨髓细胞中的Y染色体数量,进行嵌合体分析。脾细胞移植18d后,每3d观察受体小鼠的临床表现,包括体质量、体形、体位、毛发变化及生存情况,并予以评分。输注脾细胞后100d观察皮肤、肝脏、回盲端小肠靶器官的病理变化,并进行评分。结果：48只受体小鼠均进入结果分析。①嵌合体检测：6×10^7个脾细胞移植组可形成较为稳定的低水平供受者混合嵌合体;9×10^7个脾细胞移植组起初可形成较高水平的供受者混合嵌合体,随时间的推移水平降低;3×10^7个脾细胞移植组未能形成稳定的供受者混合嵌合体。②慢性移植物抗宿主病的临床及病理评分：移植物抗宿主病发生时间多集中在供鼠脾细胞输注后30～80d,临床表现以皮肤改变、体质量减轻、弓背体态为主,病理损害以皮肤、肠道及肝脏明显。与3×10^7个脾细胞移植组比较,6×10^7,9×10^7个脾细胞移植组临床和病理评分均显著升高（P〈0.05）,慢性移植物抗宿主病发生率均显著升高（P〈0.01）;后2组临床和病理各项指标之间差异均无显著性意义（P〉0.05）。结论：输注6×10^7和9×10^7个脾细胞的小鼠成功诱导出半相合异基因造血干细胞移植慢性移植物抗宿主病,其临床和病理评价体系具有较高的一致性和实用性,为进一步分析慢性移植物抗宿主病的发病机制和评价干预因素的优劣奠定实验学基础。
AIM： The effect of allogeneic stem cell transplantation （alloSCT） is limited by graft-versus-host disease （GVHD） because of differences between major and minor histocompatibility antigen. At present it is short of animal model and estimation system internally in this research. In this study, we established a mouse model and scoring system of chronic graft-versus-host disease （cGVHD） after haploidentical alloSCT. METHODS： The experiment was performed at the animal laboratory of Southern Medical University from March to August 2007. （1）Forty donor male mice （Balb/C^H-2d） of 8-10 weeks old and 18-22 g were selected. Forty-eight recipient （Balb/c× C57BL/ 6） F1^h2-d/b （CB6F1） female mice of 10-12 weeks old and 18-24 g were randomly divided into four groups （n=12）： 3 experimental groups injected with different number of spleen cells： 3×10^7, 6×10^7 and 9×10^7, respectively, and control group, All mice （SPF grade） were provided by the animal laboratory of Southern Medical University. The procedure during experiment accorded with animal ethical standards. （2）Balb/C^H-2d mice were executed and spleen cells was flushed from donor femurs and tibias with RPMI1640 and passed through sterile mesh filters to obtain single cell suspensions. Red cells were removed by Tris-NH4Cl. Spleen cells were washed and resuspended in RPMI 1640 at 1.2×10^8 L^-1 before injection. Spleen cell count was determined by trypan blue staining （95.6%）. Recipients received a single injection of 0.5 mL single cell suspensions with 3×10^7, 6×10^7, and 9×10^7, respectively, while the control group received 0.5 mL RPMI 1640 solution without cells. （3）Chromosome mosaics were assayed 2, 5, 8, and 12 weeks after injection to detect number of Y chromosome in bone marrow cells. After 18 days of transplantation, body mass, figure, cutaneous manifestation and survival of recipients mice were observed and scored every 3 days. 100 day after injection, pathology of target organs such as skin, liver, and ileal end small intestine were observed and scored. RESULTS： All 48 recipient mice were included in final analysis. （1）Recipient mice injected with 6×10^7 splenocytes resulted in a chronic disease with a low level of parental cell engraftment steadily, 9×10^7 group resulted in a chronic disease with a high level of parental cell engraftment, then decrease into a low level of parental cell engraftment, but 3×10^7 group did not result in a chronic disease with a low level of parental cell engraftment steadily. （2）According to the clinical and pathological scoring system, cGVHD occurred in three experimental groups 30-80 days after injection. Clinical manifestations referred to skin changes, decrease in body mass, hunching, and cutaneous, intestinal tract and liver lesions were predominant pathological lesions. Compared with 3×10^7 group, the clinical and pathological scores of 6×10^7 and 9×10^7 groups were significantly increased （P 〈 0.05）, and incidence of cGVHD was also significantly higher （P 〈 0.01）. But there was no significant difference between 6×10^7 and 9×10^7 groups （P 〉 0.05）. CONCLUSION： A murine model and scoring system of cGVHD after haploidentcal allo-HSCT is successfully established by injection of 6×10^7 and 9×10^7 spleen cells. The clinical and pathological evaluation system shows high consistency and practicality. It is necessary to study pathogenesis and evaluation of interventions of cGVHD.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2326-2330,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30471636)~~
