Li Zhen Yu, Wang Chun Qing, Lu Guang, Pan Xiu Ying, Xu Kai Lin
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221002, China,
Cell Biochem Biophys. 2014 Sep;70(1):115-22. doi: 10.1007/s12013-014-9866-y.
To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10(6) umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10(6) UCB-MNC and 2 × 10(7) MSC after TBI; (4) UCB+SC group, receiving 2 × 10(6) UCB-MNC and 2 × 10(6) spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10(6) UCB-MNC, 2 × 10(7) MSC and 2 × 10(6) spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II-IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I-II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.
为研究骨髓间充质干细胞（MSC）对小鼠异基因脐血移植（allo - UCBT）模型中造血恢复及急性移植物抗宿主病（GVHD）的影响。从C57/BL小鼠骨髓中获取MSC。通过流式细胞术（FCM）鉴定MSC表型，并检测其向成骨细胞和脂肪细胞分化的能力。一旦建立小鼠allo - UCBT和急性GVHD模型，将小鼠分为五组：（1）全身照射（TBI）组，每只小鼠在TBI后接受0.3 ml无菌生理盐水输注并作为对照；（2）脐血（UCB）组，TBI后接受2×10⁶个脐血单个核细胞（UCB - MNC）；（3）UCB + MSC组，TBI后接受2×10⁶个UCB - MNC和2×10⁷个MSC；（4）UCB + SC组，TBI后接受2×10⁶个UCB - MNC和2×10⁶个脾细胞；（5）UCB + SC + MSC组，TBI后接受2×10⁶个UCB - MNC、2×10⁷个MSC和2×10⁶个脾细胞。为评估造血干细胞（HSC）的植入情况，在移植后不同时间点检测白细胞、红细胞和血小板计数，并通过FCM鉴定嵌合率。采用急性GVHD临床评分、受体小鼠存活率及组织病理学分析评估MSC对急性GVHD的影响。体外成功获取MSC，FCM分析显示这些细胞CD90.2、CD44呈高阳性，CD34、CD45呈阴性，诱导后能够分化为成骨细胞和脂肪细胞。与UCB组相比，UCB + MSC小鼠的骨髓抑制持续时间较短，供体来源细胞百分比更高，可达22.87±4.3%，移植后第6天白细胞（WBC）、红细胞（RBC）和血小板计数开始增加。此外，UCB + MSC小鼠平均存活时间为25.0±10.55天，而UCB组为15.5±12.50天。UCB + SC小鼠在移植后第13天出现乏力、食欲不振、体重减轻、弓背和竖毛。约50%的小鼠出现皮肤溃疡、腹泻及其他急性GVHD表现，所有小鼠在20天内死亡。组织病理学分析证实为II - IV级GVHD表现。除短暂体重减轻外，部分UCB + SC + MSC小鼠出现弓背、竖毛、腹泻及其他急性GVHD表现。急性GVHD（I - II级或无GVHD）的UCB + SC + MSC小鼠临床评分低于UCB + SC组（P < 0.05）。骨髓MSC可促进小鼠allo - UCBT模型中的造血恢复并降低急性GVHD的发生率。
