CAJ | 학술논문

目的探讨利用犬骨髓间充质干细胞在体外诱导分化为血管内皮样细胞及平滑肌样细胞的实验方法.方法无菌条件下获得犬骨髓后采用密度梯度离心和贴壁培养的方法分离、纯化及培养犬骨髓间充质干细胞,使用流式细胞术鉴定其表面标志物.分别使用合适的培养基加入适当的诱导因子对纯化和扩增后的骨髓间充质干细胞进行诱导,使其在体外分化成血管内皮样细胞及平滑肌样细胞,然后进行鉴定.结果第3代骨髓间充质干细胞表面CD34表达阴性细胞数为99.98％;CD44表达阳性率为99.36％.利用第2代或第3代骨髓间充质干细胞,向血管内皮样细胞及平滑肌样细胞诱导分化,内皮样细胞呈“铺路石”样改变,血管性假性血友病因子(vWF)及CD31免疫荧光染色呈阳性;平滑肌样细胞具有“波峰-波谷”形状,α-肌动蛋白(α-actin)、钙调理蛋白(calponin)及平滑肌重链(SMMHC)免疫荧光染色为阳性.结论利用犬骨髓间充质干细胞,使用适当的细胞因子进行诱导,在体外可以成功地将其分化为血管内皮样细胞及平滑肌样细胞.
목적 탐토이용견골수간충질간세포재체외유도분화위혈관내피양세포급평활기양세포적실험방법.방법 무균조건하획득견골수후채용밀도제도리심화첩벽배양적방법분리、순화급배양견골수간충질간세포,사용류식세포술감정기표면표지물.분별사용합괄적배양기가입괄당적유도인자대순화화확증후적골수간충질간세포진행유도,사기재체외분화성혈관내피양세포급평활기양세포,연후진행감정.결과 제3대골수간충질간세포표면CD34표체음성세포수위99.98％;CD44표체양성솔위99.36％.이용제2대혹제3대골수간충질간세포,향혈관내피양세포급평활기양세포유도분화,내피양세포정“포로석”양개변,혈관성가성혈우병인자(vWF)급CD31면역형광염색정양성;평활기양세포구유“파봉-파곡”형상,α-기동단백(α-actin)、개조리단백(calponin)급평활기중련(SMMHC)면역형광염색위양성.결론 이용견골수간충질간세포,사용괄당적세포인자진행유도,재체외가이성공지장기분화위혈관내피양세포급평활기양세포.
Objective To investigate the experimental approach by which the vascular endothelial-like cells (ECs) and smooth muscle-like cells (SMCs) can be derived from the canine bone marrow mesenchymal stem cells (MSCs).Methods Density gradient centrifugation and adherent culture were adopted to isolate,purify and culture the canine MSCs.The surface markers of MSCs were determined by flow cytometry.Then the MSCs were induced to ECs and SMCs in appropriate medium plus inducing factors.ECs and SMCs were identified by immunofluorescence staining.Results CD34 negative and CD44 positive cells of MSCs at passage 3 account for 99.98％ and 99.34％,respectively.ECs induced from MSCs at passage 2 or 3 shows typical "cobblestone" appearance and positive immunofluorescence staining for yon Willebrand factor (vWF) and CD31.SMCs induced from MSCs at passage 2 or 3 were confirmed by their "hill and valley" morphology and positive immunofluorescence staining for α-actin,calponin and smooth muscle myosin heavy chain (SMMHC).Conclusion Canine MSCs can be induced to ECs and SMCs cells in appropriate medium plus inducing factors in vitro.