中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
7期
1271-1274,封4
,共5页
张彦男%邵增务%吴永超%王佰川%马凯歌%杨述华
張彥男%邵增務%吳永超%王佰川%馬凱歌%楊述華
장언남%소증무%오영초%왕백천%마개가%양술화
脊索细胞%骨髓间充质干细胞%髓核%椎间盘%共培养
脊索細胞%骨髓間充質榦細胞%髓覈%椎間盤%共培養
척색세포%골수간충질간세포%수핵%추간반%공배양
Notochordal cells%Bone mesenchymal stem cells%Nucleus pulposus%Intervertebral disc%Co-culture
目的 分离兔髓核脊索细胞及骨髓间充质干细胞(MSCs),通过共培养观察脊索细胞对MSCs增殖能力及细胞表型的影响.方法 4～6周龄新西兰兔4只,取胸腰段脊柱的髓核,用密度梯度离心法提取脊索细胞,同时取其股骨骨髓用Ficoll液分离得到MSCs,光镜观察脊索细胞和MSCs不同比例(1:2、1:1、2:1)共培养条件下细胞的生长,细胞计数试剂盒(CCK-8)法检测细胞增殖.脊索细胞和MSCs共培养(1:1)后行甲苯胺蓝染色及Ⅱ型胶原染色检测MSCs细胞表型的改变.对共培养后的MSCs进行相关基因表达的检测.结果 光镜下观察原代脊索细胞呈圆形或椭圆形,胞体大,细胞增殖不明显.MSCs呈梭形贴壁生长,旋涡状排列.CCK-8检测发现脊索细胞/MSCs1:1组细胞增殖明显高于其余各组.甲苯胺篮染色MSCs单独培养组呈阴性,共培养组呈阳性.Ⅱ型胶原染色MSCs单独培养组呈阴性,共培养组呈阳性.逆转录-聚合酶链反应(RT-PCR)检测发现共培养组蛋白聚糖及Ⅱ型胶原的表达分别为脊索细胞的2.00、1.35倍,而单独培养的MSCs则表达阴性.结论 在共培养条件下脊索细胞可以促进MSCs增殖,且细胞比例为1:1时更为显著；同时可以诱导其产生Ⅱ型胶原及聚集蛋白聚糖,表现出类软骨细胞表型.
目的 分離兔髓覈脊索細胞及骨髓間充質榦細胞(MSCs),通過共培養觀察脊索細胞對MSCs增殖能力及細胞錶型的影響.方法 4～6週齡新西蘭兔4隻,取胸腰段脊柱的髓覈,用密度梯度離心法提取脊索細胞,同時取其股骨骨髓用Ficoll液分離得到MSCs,光鏡觀察脊索細胞和MSCs不同比例(1:2、1:1、2:1)共培養條件下細胞的生長,細胞計數試劑盒(CCK-8)法檢測細胞增殖.脊索細胞和MSCs共培養(1:1)後行甲苯胺藍染色及Ⅱ型膠原染色檢測MSCs細胞錶型的改變.對共培養後的MSCs進行相關基因錶達的檢測.結果 光鏡下觀察原代脊索細胞呈圓形或橢圓形,胞體大,細胞增殖不明顯.MSCs呈梭形貼壁生長,鏇渦狀排列.CCK-8檢測髮現脊索細胞/MSCs1:1組細胞增殖明顯高于其餘各組.甲苯胺籃染色MSCs單獨培養組呈陰性,共培養組呈暘性.Ⅱ型膠原染色MSCs單獨培養組呈陰性,共培養組呈暘性.逆轉錄-聚閤酶鏈反應(RT-PCR)檢測髮現共培養組蛋白聚糖及Ⅱ型膠原的錶達分彆為脊索細胞的2.00、1.35倍,而單獨培養的MSCs則錶達陰性.結論 在共培養條件下脊索細胞可以促進MSCs增殖,且細胞比例為1:1時更為顯著；同時可以誘導其產生Ⅱ型膠原及聚集蛋白聚糖,錶現齣類軟骨細胞錶型.
목적 분리토수핵척색세포급골수간충질간세포(MSCs),통과공배양관찰척색세포대MSCs증식능력급세포표형적영향.방법 4～6주령신서란토4지,취흉요단척주적수핵,용밀도제도리심법제취척색세포,동시취기고골골수용Ficoll액분리득도MSCs,광경관찰척색세포화MSCs불동비례(1:2、1:1、2:1)공배양조건하세포적생장,세포계수시제합(CCK-8)법검측세포증식.척색세포화MSCs공배양(1:1)후행갑분알람염색급Ⅱ형효원염색검측MSCs세포표형적개변.대공배양후적MSCs진행상관기인표체적검측.결과 광경하관찰원대척색세포정원형혹타원형,포체대,세포증식불명현.MSCs정사형첩벽생장,선와상배렬.CCK-8검측발현척색세포/MSCs1:1조세포증식명현고우기여각조.갑분알람염색MSCs단독배양조정음성,공배양조정양성.Ⅱ형효원염색MSCs단독배양조정음성,공배양조정양성.역전록-취합매련반응(RT-PCR)검측발현공배양조단백취당급Ⅱ형효원적표체분별위척색세포적2.00、1.35배,이단독배양적MSCs칙표체음성.결론 재공배양조건하척색세포가이촉진MSCs증식,차세포비례위1:1시경위현저；동시가이유도기산생Ⅱ형효원급취집단백취당,표현출류연골세포표형.
Objective The notochordal cells ( NC ) and bone mesenchymal stem cells ( MSCs )were isolated co-cultured from New Zealand rabbit immature nucleus pulposus (NP),and the contribution of NC to proliferation and differentiation of MSCs was assessed.Methods NC were got from immature NP of 4 New Zealand rabbits (4-6 week-old) and purified by discontinuous gradient density centrifugation,and MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation.MSCs were cultured alone and co-cultured with NC ( 1:1/1:2/2:1 ).Cell proliferation was evaluated by cell counting Kit-8 ( CCK-8 ) -kit and expression levels of collagen Ⅱ and proteoglycan detected by toluidine blue and immunocytochemistry staining.The expression of collagen Ⅱ and proteoglycan mRNA was assayed.Results NCs and MSCs were isolated and purified.After co-culture,proliferation ability in the group of cell ratio at 1:1 ( NC:MSC) was increased significantly as compared with other groups.In the coculture group of MSCs and NC,the expression of collagen Ⅱ and proteoglycan was 2.0 and 1.35 times higher than in NC cultured alone.Conclusion By co-culture of 1:1,NC can stimulate proliferation of MSCs,and induce differentiation toward NP cells.
