Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (21): 3336-3341.doi: 10.3969/j.issn.2095-4344.1755
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Sun Heng, Ma Shize, Jiang Haiyue, Liu Xia, Teng Li
Revised:
2019-03-11
Online:
2019-07-28
Published:
2019-07-28
Contact:
Liu Xia, MD, Associate researcher, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China;
Teng Li, MD, Chief physician, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China
About author:
Sun Heng, Doctorate candidate, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Beijing 100043, China
Supported by:
the Medical and Health Science and Technology Innovation Project of Chinese Academy of Medical Sciences, No. 2017-I2M-1-007 (to JHY); the National Natural Science Foundation of China, No. 81471804 (to LX)
CLC Number:
Sun Heng, Ma Shize, Jiang Haiyue, Liu Xia, Teng Li. Three-dimensional dynamic culture of auricular chondrocytes with microcarriers in vitro[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3336-3341.
2.1 荧光倒置显微镜、冰冻切片DAPI染色、扫描电镜观察细胞生长情况 倒置显微镜下观察培养皿单层二维培养的细胞,第1代软骨细胞呈星形、多角形、短梭形,形态不均匀,21 d可传至第6代,软骨细胞呈长梭形,形态较均一,见图1。微载体培养时,软骨细胞可快速贴附于微载体表面,微载体静止培养21 d时软骨细胞外基质分泌增加,将微球包裹,聚集成团,而微球动态培养组的细胞外基质分泌量更多,微球之间结合更为牢固,见图2;扫描电镜下观察更为明显,微载体静止组微载体上的细胞量少,微球之间依靠细胞外基质部分相连,微球动态培养组细胞外基质将微球完全包裹,数个微球聚集成紧密的小团块。细胞膜绿色荧光染色和冰冻切片DAPI染色显示:微球中有细胞黏附,表面黏附的细胞更多。微载体动态培养组细胞数量多于微载体静止培养组,而且动态培养组细胞分布更为均匀,见图2。 2.2 增殖能力 如图3所示,培养皿单层培养2-4 d为细胞对数生长期,5 d时细胞量达到高峰;微载体动态培养组软骨细胞对数生长期延长为2-10 d,细胞数量在14 d达到高峰;微载体静止培养组细胞增殖速度缓慢。培养14 d时,培养皿单层培养细胞总量增加约3.3倍,微载体动态培养组细胞总量增加约7.3倍,微载体静止培养组细胞总量增加约2.2倍。在培养第14天,微载体动态培养组细胞数量显著高于另外2组(P < 0.001),培养皿单层培养组高于微载体静止培养组(P < 0.05)。 2.3 软骨细胞表型相关基因表达 各组软骨细胞培养 21 d时,软骨基质特异性基因ACAN和COL2相对表达量在微载体动态培养组表达最高,与其他2组比较差异有显著性意义。SOX9基因相对表达量在3组之间差异无显著性意义。COMP基因相对表达量在微载体动态培养组和微载体静止培养组之间差异有显著性意义,见图4。"
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