外周血来源间充质干细胞的成骨及成脂诱导分化

Differentiation of peripheral blood mesenchymal stem cells into adipoblasts and osteoblasts

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3cm×3cm全层皮肤,建立创伤模型。术后5d,两组大鼠腹主动脉抽血,8mL/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1×10^5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1×10^4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21d出现钙沉积,茜素红染色呈阳性;成脂诱导后14d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。

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. （1）Twenty-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. （2）After 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. （3）Colony forming efficiency （CFE） of mesenchymal stem cells was counted in every 1 × 10^5 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 ×10^4 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： （1）Observation on cell morphology： Primary culture PBMSCs presented fibroblast-like cells, （2）CFE 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. （3）Cell identification： PBMSCs were negative for CD45 and CD34, but positive for type Ⅰ collagen. （4） Differentiation 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： （1）CFE 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. （2）PBMSCs cultured in vitro can differentiate into adipocytes and osteoblasts.