CAJ | 학술논문

背景:目前培养软骨细胞多使用胎牛血清.但近年异种血清培养组织向临床应用的安全性受到质疑,因此自体血清培养越来越受到重视.目的:比较体积分数10%兔自体血清与体积分数10%胎牛血清培养兔关节软骨细胞生物学特性的差异.方法:兔自体血清培养液制备后,分离培养兔关节软骨细胞,分别在体积分数10%自体血清和体积分数10%胎牛血清中进行单层传代培养至1,3,5代,采用光镜观察细胞形态变化,绘制生长曲线评估细胞增殖速度,观察甲苯胺蓝染色、Ⅰ,Ⅱ型胶原免疫组织化学染色结果,以流式细胞仪分析细胞Ⅰ,Ⅱ型胶原以及 CD26,CD44的表达变化.结果与结论:①在细胞形态上自体血清和胎牛血清培养的软骨细胞差异不大.②自体血清培养的软骨细胞较胎牛血清培养的软骨细胞生长速度更快.③甲苯胺蓝染色结果示,无论是自体血清还是胎牛血清所培养的细胞,染色随代龄的增加逐渐变浅,细胞传至第 5代时两组几乎均无异染.对于1代和3代细胞而言,自体血清培养的软骨细胞较胎牛血清所培养的软骨细胞较为深染.④Ⅰ型胶原的表达随代数的增加而增加,而Ⅱ型胶原的表达则随代数的增加而减少.在3代时自体血清培养的软骨细胞Ⅰ型胶原的表达水平低于胎牛血清培养的软骨细胞(P <0.05);在1代和3代时自体血清培养的软骨细胞Ⅱ型胶原的表达水平高于胎牛血清培养的软骨细胞(P <0.05).⑤CD26表达呈现先升高后降低的趋势,而 CD44的表达不随传代数的增加而改变.提示同异体体积分数10%的胎牛血清相比,体积分数10%的自体血清培养的兔软骨细胞生长速度快,且在大部分指标上可以较好地保持细胞表型.
배경:목전배양연골세포다사용태우혈청.단근년이충혈청배양조직향림상응용적안전성수도질의,인차자체혈청배양월래월수도중시.목적:비교체적분수10%토자체혈청여체적분수10%태우혈청배양토관절연골세포생물학특성적차이.방법:토자체혈청배양액제비후,분리배양토관절연골세포,분별재체적분수10%자체혈청화체적분수10%태우혈청중진행단층전대배양지1,3,5대,채용광경관찰세포형태변화,회제생장곡선평고세포증식속도,관찰갑분알람염색、Ⅰ,Ⅱ형효원면역조직화학염색결과,이류식세포의분석세포Ⅰ,Ⅱ형효원이급 CD26,CD44적표체변화.결과여결론:①재세포형태상자체혈청화태우혈청배양적연골세포차이불대.②자체혈청배양적연골세포교태우혈청배양적연골세포생장속도경쾌.③갑분알람염색결과시,무론시자체혈청환시태우혈청소배양적세포,염색수대령적증가축점변천,세포전지제 5대시량조궤호균무이염.대우1대화3대세포이언,자체혈청배양적연골세포교태우혈청소배양적연골세포교위심염.④Ⅰ형효원적표체수대수적증가이증가,이Ⅱ형효원적표체칙수대수적증가이감소.재3대시자체혈청배양적연골세포Ⅰ형효원적표체수평저우태우혈청배양적연골세포(P <0.05);재1대화3대시자체혈청배양적연골세포Ⅱ형효원적표체수평고우태우혈청배양적연골세포(P <0.05).⑤CD26표체정현선승고후강저적추세,이 CD44적표체불수전대수적증가이개변.제시동이체체적분수10%적태우혈청상비,체적분수10%적자체혈청배양적토연골세포생장속도쾌,차재대부분지표상가이교호지보지세포표형.
BACKGROUND: At present, fetal bovine serum is mainly use for chondrocytes culture. However, because the security of the chondrocytes culture using heterologous serum in clinical application has been questioned in recent years, the autologous serum for chondrocytes culture has been paid more and more attention. OBJECTIVE: To compare the difference in the biological properties between rabbit articular chondrocytes cultured with 10% autologous serum and 10% fetal bovine serum in vitro. METHODS: After the culture medium of rat autologous serum was prepared, rabbit articular chondrocytes were isolated from rabbits and seeded into the medium with 10% autologous serum and 10% fetal bovine serum, respectively for monolayer subcultivation. Then, passage1, 3 and 5 cel s were chosen and cel morphological changes were observed by light microscope, meanwhile, growth curves were drawn to evaluate cel poliferation rate. Besides, biological properties of chondrocytes were observed by toluidine blue staining, type addition, the expression changes of typeⅠ,typeⅡcolagen,CD26 and CD44 were analyzed by flow cytometry. RESULTS AND CONCLUSION: (1)There was no significant difference in cel morphology between chondrocytes cultured with autologous serum and fetal bovine serum. (2)The cel growth rate of chondrocytes cultured with autologous serum was m ore rapid than that of chondrocytes cultured with fetal bovine serum. (3)Toluidine blue staining showed that both chondrocytes cultured with autologous serum and fetal bovine serum showed a passage-dependent reduction of staining. In passage 5 cel s, the two groups almost had no different dying. For passage 1 and 3 cel s, chondrocytes cultured with autologous serum showed deeper staining than fetal bovine serum. (4)Immunohistochemical staining and flow cytometry results showed that expression of type Ⅰ col agen was increased with passage, while type Ⅱ col agen expression was decreased with passage. Moreover, typeⅠcol agen expression of chondrocytes cultured with autologous serum was significantly lower than that chondrocytes cultured with fetal bovine serum at passage 3 (P < 0.05). (5)CD26 expression was increased at first and then decreased, while the expression of CD44 did not change with passage. These results suggest that compared with 10% fetal bovine serum, the growth rate of chondrocytes cultured with 10%autologous serum is more rapid, and has a better preservation of phenotype based on the results of different observations.