CAJ | 학술논문

目的为肌腱组织工程筛选出一种优化的人肌腱细胞培养基,即使在不添加胎牛血清(fetal bovine serum,FBS)的条件下,通过优化培养基构成维持其存活并分化.方法在α-MEM培养基中,加入最佳组合的生长因子(growth factors,GF),同时加入不同浓度的FBS(0、1％、5％和10％)和不同浓度的IGF-1(0、10、50 ng/ml),TGFβ-3 (0、1、10 ng/ml),用拉马拉蓝染色法检测人肌腱细胞的生存情况、增殖速度,用天狼星红定量胶原合成.结果在无FBS情况下,含有10 ng/ml TGFβ-3和50 ng/ml IGF-1两种生长因子的α-MEM培养基中培养14 d后,人肌腱细胞依旧存活(6228.68±43.87),高于其他实验组,但低于10％ FBS对照组(13 576.74±286.75,t=41.29,P＜0.05),胶原合成量[ (0.322 ±0.003) ng]明显大于未添加生长因子组(t=4.13 ～5.93,P＜0.05).结论添加50 ng/ml IGF-1和10 ng/ml TGF3-3优化的α-MEM培养基可维持人肌腱细胞存活长达14 d并促进肌腱细胞分化,而不必添加FBS.
목적 위기건조직공정사선출일충우화적인기건세포배양기,즉사재불첨가태우혈청(fetal bovine serum,FBS)적조건하,통과우화배양기구성유지기존활병분화.방법 재α-MEM배양기중,가입최가조합적생장인자(growth factors,GF),동시가입불동농도적FBS(0、1％、5％화10％)화불동농도적IGF-1(0、10、50 ng/ml),TGFβ-3 (0、1、10 ng/ml),용랍마랍람염색법검측인기건세포적생존정황、증식속도,용천랑성홍정량효원합성.결과 재무FBS정황하,함유10 ng/ml TGFβ-3화50 ng/ml IGF-1량충생장인자적α-MEM배양기중배양14 d후,인기건세포의구존활(6228.68±43.87),고우기타실험조,단저우10％ FBS대조조(13 576.74±286.75,t=41.29,P＜0.05),효원합성량[ (0.322 ±0.003) ng]명현대우미첨가생장인자조(t=4.13 ～5.93,P＜0.05).결론 첨가50 ng/ml IGF-1화10 ng/ml TGF3-3우화적α-MEM배양기가유지인기건세포존활장체14 d병촉진기건세포분화,이불필첨가FBS.
Objective To optimize the culture media by adding the growth factors required to maintain tenocytes survival and promote their differentiation without fetal bovine serum (FBS) supplementation,in order for the approach to be used for any future tendon tissue engineering.Methods The human tenocytes were cultured in α-MEM media by adding FBS at various concentrations and supplementing both insulin-like growth factor 1 ( IGF-I ) and transforming growth factor β-3 ( TGFβ-3 ).A number of growth factors were selected that could support tenocytes expansion at reduced differentiated state with the minimum FBS. By employing fractional factorial design,different treatment groups went through AlamarBlueTM tests to evaluate the cell number growth whilst collagen quantification by real time RT-PCR technique and tenocyte differentiation were also studied.Results The tenocytes cultured for 14 days with 0％ FBS,50 ng/ml IGF-1 and 10 ng/ml TGFβ-3 maintained survival over 14 days,the Cell count were 6228.68 ± 43.87.They were higher than the other experimental groups,but less than 10％ FBS control group ( 13 576.74 ±286.75,t =41.29,P ＜0.05).The tenocytes cultured in the treated group also showed enhanced collagen synthesis ((0.322 ±0.003) ng,t =4.13-5.93,P ＜0.05). Conclusion These findings have shown for the first time that human tenocytes could be maintained survival for a long period of time in the culture media without FBS,having this approach a suitable one for tendon tissue engineering.