骨髓腔输注间充质干细胞对骨髓移植后造血重建和移植物抗宿主病作用的实验研究

Experiment study of efficacy on hematopoietic reconstitution and GVHD prophylaxis after mesenchemal cell infused by intro-bone marrow cavity or introveinous in rat BMT models

目的比较骨髓腔内（IBM）及静脉内（IV）输注异基因间充质干细胞（MSC）对大鼠骨髓移植（BMT）模型的骨髓造血、MSC重建和移植物抗宿主病（GVHD）的影响，优化MSC输注途径及探讨MSC促进造血的机制。方法①MSC体内动力学示踪。②建立大鼠BMT模型。③实验分组：IBM组[MSC（IBM）＋骨髓有核细胞（IV）]；IV组[MSC（IV）＋骨髓有核细胞（IV）]；骨髓移植（BMT）组；单纯MSC组；照射对照组；空白对照组。④移植后观察受体存活、外周血常规结果及植入证据、骨髓MSC、CFU-Mix恢复及GVHD。结果①IBM途径输注MSC主要分布于骨髓。②MSC与骨髓共移植可以提高大鼠存活率：照射对照组、单纯MSC输注组均在预处理后2周内全部死亡；BMT组2周内存活率为70％；IV存活率为80％；IBM组存活率为100％。③MSC可促进BMT大鼠外周血象恢复：移植后第14天（＋14天）移植组白细胞计数均明显高于BMT组，IBM组又明显高于IV组，＋21天IBM组三系均恢复，IV组＋21天白细胞计数恢复，血红蛋白浓度和血小板计数至＋30天恢复正常；BMT组三系恢复延迟约2周。④MSC促进BMT受体骨髓MSC恢复和造血重建：＋21天和＋30天共移植组CFU-Mix和MSC克隆生长恢复均较BMT组快（P＜0．05）；IBM组优于IV组（P＜0．05）。⑤植入证据的检测：BMT及共移植组存活大鼠外周血均检测到F344大鼠供体抗原RT1A’阳性细胞，比例＞90％。⑥GVHD发生率：BMT组GVHD发生率为42％；共移植组无明显GVHD表现。结论IBM输注MSC优先定位于骨髓、极少丢失于非造血器官；输注MSC可促进BMT后造血恢复及提高受体生存率；MSC促进BMT受体骨髓MSC及造血干细胞恢复，缩短骨髓抑制期，并降低GVHD；IBM输注途径效果优于IV途径。

Objective To observe the in vivo distribution of mesenchemal stem cells （MSCs） after administrated by intro-bone marrow（IBM） or introveinous（ IV）, and compare the effects on hematopoiesis reconstitution and GVHD in rat BMT models. Methods （ 1 ） MSCs from male Wistar rats marked with CFSE were injected into the bone marrow cavity（IBM） or the vein（IV） of recipient rats, and observed the distribution of MSCs in vivo. （2） Allogeneic BMT model of Fischer344 rats （ RT1A^1 ） to Wistar rats （ RT1A^u ） was established. The recipient rats were exposed to 8 Gy of gamma irradiation 1 day before transplantation. The 6 groups were ①IBM group [ IBM-injection of MSCs ＋ IV-injection of bone marrow cells（BMC） ] ; ②IV group（ IV-injectian of MSCs （IV） ＋ IV-injection of BMC） ; ③BMT group（ only IV-injection of BMC） ; ④ MSCs control group（ only IV-injection of MSC） ; ⑤normal control group and ⑥irradiation control group. Results ①After IV-injection, large numbers of the MSCs lodged in lungs while small numbers in the peripheral blood, liver, thymus and spleen, and a few marked MSCs could be seen in bone marrow. After IBM injection, most cells distributed in long bones and those lungs were less than that in IV group. ②Co-transplantation of MSCs （IBM/IV） could accelerate the recovery of hematopoiesiS, including the recovery of WBC,hemoglobin and platelet, and in IBM-injection was more effective in the recovery of hematopoiesis than that in IV group. ③Incidence rate of GVHD in BMT group was 42% （3/7）, and no GVHD occurred in co-transplantation groups. ④ Recovery of CFU-Mix and CFU-MSCs could be seen at 21th and 30th day after trans- plantation in co-transplantation groups, and IBM-injection was more effective than IV-injection. Conclusion ① IBM-injection results in most MSCs distributed in long bones. ②MSCs improve the survival rate after BMT. ③Co-transplantation of MSCs accelerates the recovery of hematopoiesis and reduces the morbidity of GVHD. ④MSC promotes reconstitution of hematopoietic cells and bone marrow MSCs in recipient rates and the effects of MSCs administrated via IBM is more effective than via IV.