自体血清与胎牛血清培养兔关节软骨细胞的生物学特性

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。

Biological properties of rat articular chondrocytes cultured with autologous serum versus fetal bovine serum

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 cells were chosen and cell morphological changes were observed by light microscope,meanwhile,growth curves were drawn to evaluate cell poliferation rate.Besides,biological properties of chondrocytes were observed by toluidine blue staining,type Ⅰ and typeⅠ collagen immunohistochemical staining.In addition,the expression changes of type Ⅰ,type Ⅰ collagen,CD26 and CD44 were analyzed by flow cytometry.RESULTS AND CONCLUSION：（1）There was no significant difference in cell morphology between chondrocytes cultured with autologous serum and fetal bovine serum.（2）The cell 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 cells,the two groups almost had no different dying.For passage 1 and 3 cells,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 collagen was increased with passage,while type Ⅱ collagen expression was decreased with passage.Moreover,typeⅠcollagen expression of chondrocytes cultured with autologous serum was significantly lower than that chondrocytes cultured with fetal bovine serum at passage 3（P0.05）.（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.