小鼠半相合移植物抗宿主病体内、外实验体系的建立及应用  

Establishment and Application of T Cells and Mice Model in Screening Drugs for Prevention and Treatment of Graft Versus Host Disease

作　　者：林筱洁[1] 高瑞兰[1] 倪桂宝[1] 尹利明[1] 沃立科[1] 董原[1] 赵燕娜[1] 王潇[1] 陈小红[1] 

机构地区：[1]浙江中医药大学附属第一医院血液病研究所,杭州310006

出　　处：《浙江中医药大学学报》2013年第8期945-950,955,共7页Journal of Zhejiang Chinese Medical University

基　　金：浙江省自然科学基金(Y207632)~~

摘　　要：[目的]建立观察小鼠移植物抗宿主病(Graft Versus Host Disease,GVHD)的体、内外实验体系。[方法]体外实验体系包括MTT法观察单向混合淋巴反应(MLR)中供鼠淋巴细胞增殖;采用PHA活化供鼠T淋巴细胞,Annexin V/PI双染法检测T细胞凋亡率,观察细胞周期变化及不同时间的CD25和CD69表达率。选择经典GVHD防治药物环孢菌素A(cyclosporin A,CSA)和地塞米松(dexamethasone,Dexa)验证体外实验体系的有效性。以CB6F1为受鼠,60Co全身照射清髓,输入供鼠的骨髓和脾细胞,建立稳定的小鼠半相合骨髓移植GVHD模型,为体内实验体系,观察GVHD的程度、生存期及CSA和Dexa的疗效。[结果]CSA和Dexa能显著抑制单向MLR中供鼠效应T淋巴细胞增殖,诱导PHA活化后T淋巴细胞凋亡率增加。CSA和Dexa对T细胞的细胞周期影响包括亚二倍体凋亡峰的比例明显增加、G0/G1期比例增加和S期比例减少。CSA和Dexa还能降低GVHD同种反应T细胞的标志CD25和CD69的表达。建立稳定的小鼠半相合骨髓移植GVHD模型,CSA和Dexa均能减轻GVHD的程度,延长生存期,以CSA为佳。[结论]形成体外观察供鼠GVHD同种反应T细胞增殖、活化的实验体系,建立稳定的小鼠GVHD模型,采用CSA和Dexa验证了其药物筛选的有效性。[Objective]To establish T cell and mice model in screening effective drug for graft versus host disease（GVHD）. [Methods]The lymphocytes from the spleen of donor mice were prepared by routine method. The proliferation of spleen lymphocyte in the one-way mixed lymphocyte reaction（MLR） was determined by MTT method in vitro. The spleen lymphocytes from donor mice were stimulated by PHA, then marked with CD3 antibody, followed by Annexin V and PI stain, and detected by Flow Cytometry, CD3＋ T lymphocytes were first selected and Annexin V＋/PI- apoptosis cells rate was deter- mined. The cell cycle of CD3~ T lymphocytes and their CD25 and CD69 expression rate after PHA activation were detected by Flow Cytometry. Then classic GVHD prevention drug cyclosporin A（CSA） and dexamethasone（Dexa） were chosen to validate this drug screening system of mouse T lymphocyte proliferation and activation in vitro. The stable mice GVHD model after half-compatible hematopoietic stem cell transplantation was established, the CB6F1 recipients mice were 6~co irradiated all over the body, the bone marrow and splenocytes from donor mice were injected via tail vain within 4 hour a^er irradiation. The degree of GVHD, life span and the curative effect of CSA and Dexa were observed. [Results] CSA and Dexa can significantly inhibit the proliferation of lymphocyte from donor mice in one-way MLR, CSA and Dexa induce the apoptosis of T lymphocyte after PHA stimulation. The ef- fect of CSA and Dexa on cell cycles of T lymphocytes includes that the proportion of sub-diploid apoptosis peak significandy increases, the rates of GO/ G1 phase raise, while the proportion of S phase decreases. CSA and Dexa can also decrease the expression rate of CD25 and CD69, which are the markers of T cells response for GVHD. The stable mice GVHD model after half-compatible hematopoietic stem cell transplantation is established, CSA and Dexa can reduce the degree of GVHD, prolong the survival period, CSA is better. [Conclusion] The proliferation and activa

关 键 词：移植物抗宿主病 骨髓移植 T细胞凋亡 T细胞周期 CD25 CD69 

分 类 号：R331[医药卫生—人体生理学]