CAJ | 학술논문

[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。
[목적]건립관찰소서이식물항숙주병（Graft Versus Host Disease，GVHD）적체、내외실험체계。[방법]체외실험체계포괄MTT법관찰단향혼합림파반응（MLR）중공서림파세포증식；채용PHA활화공서T림파세포，Annexin V/PI쌍염법검측T세포조망솔，관찰세포주기변화급불동시간적CD25화CD69표체솔。선택경전GVHD방치약물배포균소A（cyclosporin A,CSA）화지새미송（dexamethasone, Dexa）험증체외실험체계적유효성。이CB6F1위수서，60Co전신조사청수，수입공서적골수화비세포，건립은정적소서반상합골수이식GVHD모형，위체내실험체계，관찰GVHD적정도、생존기급CSA화Dexa적료효。[결과] CSA화Dexa능현저억제단향MLR중공서효응T림파세포증식，유도PHA활화후T림파세포조망솔증가。CSA화Dexa대T세포적세포주기영향포괄아이배체조망봉적비례명현증가、G0/G1기비례증가화S기비례감소。CSA화Dexa환능강저GVHD동충반응T세포적표지CD25화CD69적표체。건립은정적소서반상합골수이식GVHD모형，CSA화Dexa균능감경GVHD적정도，연장생존기，이CSA위가。[결론]형성체외관찰공서GVHD동충반응T세포증식、활화적실험체계，건립은정적소서GVHD모형，채용CSA화Dexa험증료기약물사선적유효성。
[Objective]To establish T cel 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, fol owed by Annexin V and PI stain, and detected by Flow Cytometry, CD3+T lymphocytes were first selected and Annexin V+/PI- apoptosis cel s rate was deter-mined. The cel 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 cel transplantation was established, the CB6F1 recipients mice were 60co irradiated al over the body, the bone marrow and splenocytes from donor mice were injected via tail vain within 4 hour after 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 cel cycles of T lymphocytes includes that the proportion of sub-diploid apoptosis peak significantly increases, the rates of G0/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 cel s response for GVHD. The stable mice GVHD model after half-compatible hematopoietic stem cel transplantation is established, CSA and Dexa can reduce the degree of GVHD, prolong the survival period, CSA is better.[Conclusion] The proliferation and activation of mouse T lymphocyte system for drug screening in vitro and stable mice GVHD model are successful y established, and validated using CSA and Dexa.