【作者】 王斌；

【导师】 王健民；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 目的研究白血病细胞上B7-H2的表达与异基因造血干细胞移植GVL效应的关系。方法对47例初发的急性白血病患者,应用流式细胞术观察其白血病细胞上B7H2的表达情况,分析其与预后因素的关系。通过流式细胞术及实时定量PCR的方法检测小鼠白血病细胞上B7-H2的表达及其胞内mRNA水平；选用小鼠淋巴瘤细胞株A20作为靶细胞,用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记靶细胞,再用碘化丙碇(PI)标记DNA筛选细胞膜已破坏的靶细胞。效靶比为50：1-10：1,共孵育时间为12小时,流式细胞仪收集1000个靶细胞并测定被杀细胞的百分率。检测其对异基因靶细胞的细胞毒作用的影响。将带有小鼠B7H2基因shRNA片断的质粒载体PGCsi3.0通过脂质体转入高表达B7H2的小鼠淋巴瘤细胞株HBA20。以C57BL/6雄性小鼠为供体,BALB/C雌性小鼠为受体,BALB/C来源的HBA20细胞8×104／只为白血病细胞,同时输注异基因的C57小鼠脾细胞2×106/只,建立小鼠GVL模型,在此基础上输入转有shRNA质粒的小鼠HBA20细胞,观察B7-H2基因沉默后对异基因造血干细胞移植GVL效应的影响。结果B7H2在急性髓细胞白血病(n=29)和急性淋巴细胞白血病(n=19)的阳性率分别为25%和31.42%。低危组与中／高危组差异具有统计学意义(P<0.05)。在效靶比为10：1、20：1、30：1时,阻断肿瘤细胞上的B7-H2,可以明显提高效应细胞对肿瘤细胞的杀伤作用,两组的差别具有统计学意义(P值分别为0.0302、0.0496、0.0152)；随着效靶比例的进一步升高,至40:1、50：1时,阻断肿瘤细胞上的B7-H2不能有效地提高效应细胞对肿瘤细胞的杀伤作用,两组的差别没有统计学意义(P值分别为0.6972、0.4021)。体内实验显示,E组(输转染空白质粒的HBA20细胞及脾细胞)与F组(输转染shRNA质粒的HBA20细胞及脾细胞)差异有统计学意义P=0.0005。结论人类白血病细胞有不同程度的B7H2的表达,其与预后因素相关。体外实验表明,在适当的效、靶比时,封闭白血病细胞上B7-H2,可以有效地提高异基因靶细胞的细胞毒作用。体内实验显示,小鼠淋巴瘤白血病细胞株A20进行B7-H2基因沉默后可以增强异基因造血干细胞移植GVL效应,延长生存时间。更多还原

【Abstract】 Objective The aim of this study was to examine the effect of B7H2 gene silencing in mice A20 cell on the graft-versus-leukemia (GVL) reaction after allogeneic bone marrow transplantation. Methods Forty-seven new patients with adult de novo acute leukemia, who were diagnosed according to the French-American-British criteria, were the subjects of this study. We choose A20 as target cell. The test principle is based on target cell labeling with CFSE and subsequent DNA-labelling with PI for identification of target cells with compromised cell membranes. Effectors and targets were incubated at E/T ratios from 50:1 to 10 : 1 for 12 h, collect 1000 targetevents and analyze specific cytotoxicity. We introduced shRNA-pGCsi which could inhibite the expression of B7H2 in A20 cells and plasmid vector pGCsi into A20 cell line using lipofectamine method respectively. Then we infused them into mice intravenously to erect the model of GVL respectively to examine the effect of B7H2 gene silencing in mice A20 cell on the graft-versus-leukemia (GVL) reaction after allogeneic bone marrow transplantation. Result The cells from a substantial number of acute leukemia cases expressed B7-H2 molecules [percentages of B7-H2 positive cases in AML and ALL were 25%(n= 29) and 31%(n= 19). Mean percentages of B7-H2-positive cells in patients of high risk was significantly higher than those of low risk[Mean percentages were 4%(n= 5) and 15%(n=7), respectively].We observed that B7H2 blocked A20 cells obtained more cytolytic activity than B7H2 positive A20 cells at E/T ratios 30:1,20:1 and 10:1 [P=0.0062, 0.0287,0.0462, respectively]. B7H2 blocked A20 cells obtained no more cytolytic activity than B7H2 positive A20 cells at E/T ratios 40:1 and 50:1 [P=0.1318,0.2804, respectively].The difference between GVL group one and GVL group two was statistically significant(P=0.005), but no difference was observed among Leakemia group (P=0.640). Conclusion Our results indicate the expression of functional B7-H2 molecules, and this molecules may facilitate progression of acute leukemia. These results indicate that B7H2 blocked A20 cells obtained more cytolytic activity than B7H2 positive A20 cells at proper E/T ratios. Mice that received B7H2 gene silenced A20 cells demonstrated delayed mortality and delayed tumor progression.更多还原