犬骨髓间充质干细胞体外诱导分化的平滑肌细胞的组织培养及鉴定

目的：应用条件培养基体外诱导成年犬骨髓间充质干细胞分化为平滑肌细胞，探讨其作为构建组织工程血管种子细胞的可行性。方法：应用DMEM添加PDGF-BB和维生素C(VIT-C)体外培养通过密度梯度离心法得到的骨髓单核细胞，通过形态学观察鉴定培养的细胞，采用免疫荧光法检测4～6代细胞中平滑肌细胞特异性标记α-肌动蛋白（α-SMA）和肌球蛋白重链SMMHC，应用流式细胞术检测第5、6代细胞中平滑肌细胞的阳性率。结果：犬骨髓间充质干细胞在条件培养基的诱导下稳定传代至第6代后达到种子细胞的种植数量10⁷～10⁸，单独DMEM培养需要42　d，添加PDGF-BB/VIT-C后时间减少为33　d。细胞免疫荧光结果提示,大部分细胞α-SMA阳性、SMMHC阴性;流式细胞术结果显示,第5、6代细胞的α-SMA阳性率分别为57.8%和66.8%。结论：体外条件培养基能诱导犬骨髓间充质干细胞增殖分化为平滑肌细胞，并可作为组织工程血管构建的种子细胞。

Culture and identification of smooth muscle cells induced with canine bone marrow-derived mesenchymal stem cells in vitro

Objective To explore the possibility of differentiation of canine bone marrow-derived mesenchymal stem cells(BMSCs) into smooth muscle cells(SMCs) and the potential of using these SMCs as cell sources for engineering of blood vessel construction.Methods Canine BMSCs were isolated by density gradient centrifugation and cultivated in DMEM supplemented with PDGF-BB and vitamin C(VIT-C).The phenotypic characteristics of BMSCs were identified by morphological observation,α-SMA and SMMHC in SMCs at passage 4-6 were analyzed by immunofluorescent staining,and the positive rates of SMCs at passage 5,6 were detected by flow cytometry.Results The BMSCs cultivated in the conditioned medium for SMCs showed SMC-like morphology:they displayed spindle-shape in morphology and were positive for α-SMA but negative for SMMHC.42 d and 33 d were needed to obtain 107-108 seeding cells without or with PDGF-BB/VIT-C in culture medium respectively.With the subculture,the percentage of α-SMA positive cells increased from 57.8% at passage 5 to 66.8% at passage 6,suggesting under aforementioned cultured conditions,more BMSCs turned into SMCs.Conclusion BMSCs can be differentiated into SMCs under appropriate culture conditions,suggesting the potentiality of using these SMCs as cell sources for tissue engineering of blood vessel construction.