课题背景：：文章针对皮肤损伤与非损伤条件下外周血来源间充质干细胞的分离培养及成骨成脂分化进行研究，具理论意义和应用前景。但在实验设计上，外周血来源间充质干细胞采用密度梯度离心法，而作为对照的骨髓间充质干细胞采用全骨髓法。目前间充质干细胞的分离方法有多种，被不同实验室根据经验和条件所采用，但在同一个对照研究中实验组和对照组应采用统一方法，以更具可比性，特别是在混合细胞群这一本身学术争议较大的领域时更应注意。

创新要点：正常情况下，外周血来源间充质干细胞在血液中的含量较少，实验采取致大鼠皮肤损伤后抽血分离外周血来源间充质干细胞，克隆数量明显增加，提高了分离培养效率。

偏倚或不足：①实验从SD大鼠获得外周血来源间充质干细胞，因实际条件限制没有以人的血样为材料分离外周血来源间充质干细胞。②采用密度梯度离心法分离培养的外周血来源间充质干细胞扩增能力很弱，其是否与骨髓来源间充质干细胞一样也能够参与皮肤的创伤修复还有待深入探讨。

摘要
目的：目前对于是否存在外周血来源间充质干细胞备受争议。实验拟进一步探讨在皮肤损伤与非损伤条件下，外周血来源间充质干细胞的分离培养及其成骨、成脂多向分化特性。
方法：实验于1996-08/1997-11在解放军第四军医大学组织工程研发中心完成。①动物：清洁级SD大鼠24只，随机数字表法分为正常组、创面组，12只/组，实验过程中对动物的处置符合动物伦理学标准。②实验方法：创面组大鼠麻醉后，背部切去3 cm×3 cm全层皮肤，建立创伤模型。术后5 d，两组大鼠腹主动脉抽血， 8 mL/只，加入含体积分数为0.1胎牛血清的α-MEM培养基，密度梯度法分离培养外周血来源间充质干细胞。取大鼠双侧股骨、胫骨，全骨髓法分离培养骨髓来源间充质干细胞作为对照。③实验评估：培养至第12天，计数原代培养每1×105个外周血单个核细胞所形成的间充质干细胞克隆数。取第3代外周血间充质干细胞，行CD45、CD34、I型胶原免疫组织化学鉴定细胞表型；按1×104密度接种于12孔板，细胞贴壁后分别换用成骨、成脂诱导液培养，以茜素红、油红染色检测其分化特性。
结果：①细胞形态观察：原代培养的外周血来源间充质干细胞呈成纤维样。②克隆形成：外周血来源间充质干细胞形成的克隆数仅为骨髓来源间充质干细胞的1/6~1/8，创面组外周血间充质干细胞形成的克隆数是正常组3~4倍。③细胞表型鉴定：外周血来源间充干细胞CD45、CD34呈阴性，I型胶原呈阳性。④成骨及成脂诱导：外周血间充质干细胞成骨诱导后21 d出现钙沉积，茜素红染色呈阳性；成脂诱导后14 d有少量脂滴出现，油红染色为阳性。
结论：①皮肤损伤状态下外周血间充质干细胞克隆数量是正常条件的3~4倍，但仍远低于骨髓来源间充质干细胞的克隆能力。②体外分离培养的外周血间充质干细胞具备成骨、成脂分化特性。
关键词：间充质干细胞；外周血；创伤；分离培养；诱导

徐军军，胡刚，邹运动，刘鹏，李裕强，金岩.外周血来源间充质干细胞的成骨及成脂诱导分化[J].中国组织工程研究与临床康复，2008，12(16):3057-3060 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3057(ps).pdf]

Differentiation of peripheral blood mesenchymal stem cells into adipoblasts and osteoblasts

Abstract

AIM：The existence of peripheral blood mesenchymal stem cells (PBMSCs) is under debate. This study aimed to investigate the isolation, culture and differentiation into adipoblasts and osteoblasts of PBMSCs under wound and non-wound conditions.
METHODS: Experiments were performed at the Research and Development Center for Tissue Engineering of Fourth Military Medical University of Chinese PLA from August 1996 to November 1997. ①Twenty-four clean Sprague-Dawley rats were randomly divided into a normal group and a wound group, with 12 rats in either group. Animal interventions in the experiment met the Animal Ethical Standards. ②After anesthesia, 3 cm×3 cm full-thickness skins were cut on the back of rats in the wound group. At day 5 after surgery, peripheral blood (8 mL each) was collected from the aorta ventralis of rats in both groups. Mesenchymal stem cells were harvested from peripheral blood by density gradient centrifugation in α-MEM media containing fetal bovine serum of 0.1 volume fraction. Bone marrow mesenchymal stem cells (BMSCs) were harvested from the femur and tibia of rats by whole bone marrow method as controls. ③Colony forming efficiency (CFE) of mesenchymal stem cells was counted in every 1×105 peripheral blood mononuclear cells at day 12. Third passage of PBMSCs were detected with antibody CD45, CD34 and type Ⅰ collagen by immunohistochemical staining. The third passage of PBMSCs inoculated in a 12-well plate at 1×104 were induced with the adipocyte and osteoblast inductor after adherence. Alizarin Red S staining and Oil-Red-O staining were employed to measure the differentiation of PBMSCs.
RESULTS: ①Observation on cell morphology: Primary culture PBMSCs presented fibroblast-like cells. ②CFE counting: CFE of BMSCs was 6-8 times more than PBMSCs, and the CFE of PBMSCs from rats in the wound group was 3-4 times more than in the normal group. ③Cell identification: PBMSCs were negative for CD45 and CD34, but positive for type Ⅰ collagen. ④Differentiation into adipocytes and osteoblasts: At day 21 after differentiation of PBMSCs into osteoblasts, calcium deposition was detected by Alizarin Red S (positive staining). At day 14 after differentiation into adipocytes, a few lipid droplets were detected by Oil red-O (positive staining).
CONCLUSION: ①CFE of PBMSCs from rats in the wound group is 3 to 4 times more than in the normal group. However, the CFE is much less than BMSCs. ②PBMSCs cultured in vitro can differentiate into adipocytes and osteoblasts.

Xu JJ, Hu G, Zou YD, Liu P, Li YQ, Jin Y. Differentiation of peripheral blood mesenchymal stem cells into adipoblasts and osteoblastsDifferentiation of peripheral blood mesenchymal stem cells into adipocytes and osteoblasts. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3057-3060(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3057(ps).pdf]