慢病毒载体介导单纯疱疹病毒胸苷激酶／更昔洛韦系统抑制移植物抗宿主病的实验研究

Experimental study of the inhibiting effect of the lentiviral vector mediated herpes simplex virus-thymidine kinase/ganciclovir on GVHD

目的研究慢病毒载体介导单纯疱疹病毒胸苷激酶／更昔洛韦（HSV-TK／GCV）系统对小鼠异基因骨髓移植（allo—BMT）后移植物抗宿主病（GVHD）的防治作用。方法将转染HSV-TK基因的慢病毒感染供鼠（C57BL／6小鼠）脾脏淋巴细胞，将感染后的淋巴细胞与供鼠骨髓细胞混合，移植给经。Co1射线照射后的受鼠（BALB／c小鼠）。分别于移植后当天、移植后7天（＋7天）和＋12天腹腔注射GCV25mg／kg×7d，观察受鼠生存期、GVHD发生率及严重程度、T细胞亚群（CD3、CD4、CD8）及异基因嵌合等指标。结果HSV-TK／GCV使用0天、＋7天、＋12天组受鼠生存时间分别为（30．10±5．21）d、（36．40±5．28）d、（28．20±4．82）d，三组生存时间均较移植对照组[（15．10±0．43）d]明显延长（P〈0．05）；HSV-TK／GCV＋7天组小鼠50d存活率达60％，高于HSV-TK／GCV0天（40％）和＋12天组（30％）（P〉0．05）。对照组小鼠全部发生Ⅲ～Ⅳ级GVHD，实验组死亡小鼠有Ⅱ～Ⅲ级GVHD病理学改变，而长期生存小鼠仅出现Ⅰ～Ⅱ级GVHD。在＋5，＋10，＋15d，3个时间点实验组小鼠CD4^＋细胞明显高于对照组（P〈0．05），CD8^＋细胞均低于对照组（P〈0．05）。＋30天受鼠异基因嵌合率为100％。结论慢病毒载体介导的HSV-TK／GCV系统能有效控制小鼠allo—BMT后GVHD；＋7天外周血白细胞数开始回升时用GCV控制GVHD效果最佳。

Objective To study the effect of lentiviral vector mediated herpes simplex virus-thymidine kinase/ganciclovir （HSV-TK/GCV） on graft- remus-host disease （GVHD） after allogeneic bone mar- row transplantation （allo- BMT） in mice. Methods Donor splenic lymphocytes from C57BL/6 which were infected by lentiviral vectors carrying HSV-TK were transplanted into ^60Coγ ray irradiated recipient mice with donor bone marrow cells. GCV 25 mg·kg^-1 · d^-1 was administered in 3 groups on day 0, ＋7, ＋ 12 respectively after transplant for 7 days by intraperitoneal injection. Survival time, severity of GVHD, incidence of GVHD, T lympbocytes immune reconstruction and of allogeneic chimerism ratio were detected after allo- BMT. Results The average survival times for GCV 0 day, ＋7 day and ＋ 12 day group were （30. 10 ± 5.21 ） d, （36.40 ± 5.28 ） d and （ 28.20 ± 4.82 ） d respectively, being significantly longer than that in the control group [（15.10±0.43） d] （P 〈 0.05）. The 50 d-survival rate for TK/GCV ＋7 day group was 60%. While for 0 day and ＋ 12 day group was 40% and 30% respectively. The incidence of grade Ⅲ～Ⅳ GVHD in the control group was 100% , and the dead mice in experimental groups showed pathological chan- ges of Ⅱ～Ⅲ GVHD. Long-term alive recipient mice only developed grade Ⅰ～Ⅱ GVHD after allo-BMT. The number of CD4^＋ lymphocytes in experimental groups was higher than that in control group （ P 〈 0.05 ）, but CD8^＋ lympbocytes was lower on day ＋ 5, ＋ 10, ＋ 15 day （P 〈 0.05 ）. Allogeneic chimerism rate of recipient mice on ＋ 30 d was 100%. Conclusions HSV-TK/GCV induced by the lentiviral vectors has a deftnite effect in prevention of GVHD after allo-BMT. GCV adminstrated from 7 days pest-transplantation showed the best effects.