CAJ | 학술논문

背景:间充质干细胞不仅自身免疫原性弱,还可以调节细胞免疫功能,减轻移植物排斥反应,在组织工程中具有良好的应用前景.但骨髓中间充质干细胞含量稀少,约占单个核细胞的十万分之一到百万分之一.目的:建立一种分离、培养扩增兔骨髓间充质干细胞的方法,观察体外培养骨髓间充质干细胞的生长特性,及其潜在的诱导分化能力.方法:采用灌流法获取兔胫骨骨髓,密度梯度离心法联合贴壁培养法体外纯化扩增,相差显微镜观察其形态学特点,MTT法测定绘制传代骨髓间充质干细胞生长曲线,经成骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,0.1 μmol/L地塞米松,200 μmol/L抗坏血酸,10 mmol/L β-甘油磷酸钠)、成脂诱导液(L-DMEN/F12,体积分数为10%胎牛血清,1 μmol/L地塞米松,200 μmol/L吲哚美辛,0.5 mmol/L IBMX,10 mg/L胰岛素)、成软骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,10 μg/L转化生长因子β1,0.1 μmol/L地塞米松,50 μmol/L抗坏血酸,6.25 mg/L胰岛素)体外诱导骨髓间充质干细胞向成骨细胞、脂肪细胞及软骨细胞分化,并分别经碱性磷酸酶染色、油红O染色和甲苯胺蓝染色法鉴定.结果与结论:通过密度梯度离心法联合贴壁培养法可在体外大量扩增、纯化骨髓间充质干细胞,所获细胞具有高度自我更新能力和多向分化潜能,原代及传代骨髓间充质干细胞为梭形.经成骨细胞诱导,细胞碱性磷酸酶染色阳性;经成脂肪细胞诱导,细胞内出现红色脂滴,经成软骨细胞诱导,甲苯胺蓝染色阳性.
배경:간충질간세포불부자신면역원성약,환가이조절세포면역공능,감경이식물배척반응,재조직공정중구유량호적응용전경.단골수중간충질간세포함량희소,약점단개핵세포적십만분지일도백만분지일.목적:건립일충분리、배양확증토골수간충질간세포적방법,관찰체외배양골수간충질간세포적생장특성,급기잠재적유도분화능력.방법:채용관류법획취토경골골수,밀도제도리심법연합첩벽배양법체외순화확증,상차현미경관찰기형태학특점,MTT법측정회제전대골수간충질간세포생장곡선,경성골유도액(L-DMEN/F12,체적분수위10%태우혈청,0.1 μmol/L지새미송,200 μmol/L항배혈산,10 mmol/L β-감유린산납)、성지유도액(L-DMEN/F12,체적분수위10%태우혈청,1 μmol/L지새미송,200 μmol/L신타미신,0.5 mmol/L IBMX,10 mg/L이도소)、성연골유도액(L-DMEN/F12,체적분수위10%태우혈청,10 μg/L전화생장인자β1,0.1 μmol/L지새미송,50 μmol/L항배혈산,6.25 mg/L이도소)체외유도골수간충질간세포향성골세포、지방세포급연골세포분화,병분별경감성린산매염색、유홍O염색화갑분알람염색법감정.결과여결론:통과밀도제도리심법연합첩벽배양법가재체외대량확증、순화골수간충질간세포,소획세포구유고도자아경신능력화다향분화잠능,원대급전대골수간충질간세포위사형.경성골세포유도,세포감성린산매염색양성;경성지방세포유도,세포내출현홍색지적,경성연골세포유도,갑분알람염색양성.
BACKGROUND: Mesenchymal stem cells (MSCs), with low immunogenicity, can regulate cellular immunity and mitigate graft rejection, which has a good prospect in tissue engineering. However, it is rarely present in bone marrow. OBJECTIVE: To explore an isolation and culture method of the rabbit bone marrow-derived MSCs, to observe the biological characteristics and differentiation potential of bone marrow-derived MSCs.METHODS: MSCs were isolated from rabbit tibia bone marrow by combination of gradient centrifugation and different adherent method, then proliferation in vitro. Morphology was examined by phase contrast microscopy, and the growth curve of cultured MSCs was drawn via MTT results. MSCs were treated with osteogenetic inductor (L-DMEM/F12, 10% fetal bovine serum, 0.1 μmol/L dexamethasone, 200 μmol/L vitamin C, 10 mmol/L β-phosphoglycerol), adipose inductor (L-DMEM/F12, 10% FBS, 1 μmol/L dexamethasone, 200 μmol/L antifani, 0.5 mmol/L IBMX, 10 μg/mL insulin), and chondrocytes inductor (L-DMEM/F12, 10% FBS, 10 μg/L TGF-β1, 0.1 μmol/L dexamethasone, 50 μmol/L vitamin C, 6.25 mg/L insulin) to differentiated into osteoblast, dipocytes and chondrocytes. And the differentiated cells were identified by alkaline phosphatase staining, oil red O staining, and toluidine blue staining, respectively.RESULTS AND CONCLUSION: Bone marrow-derived MSCs can be isolated and cultured by the combination of gradient centrifugation and different adherent method in vitro, which have the better potentiality of proliferation and multi-directional differentiation. Mostly of the primary and passaged cells were spindle-shaped. After osteogenetic induction, cells were positive to alkaline phosphatase staining. Oil red O staining showed that red lipid droplet existed in adipose cells, and toluidine blue staining showed that toluidine blue was positive after chondrocytes induction.