| 刘鑫成,孟星星,祝 瑞,张煜珅,成西侠,范宏斌.使用透明质酸钠复合海藻酸钠水凝胶球3D培养软骨细胞抑制细胞去分化的研究[J].现代生物医学进展英文版,2017,17(16):3049-3053. |
| 使用透明质酸钠复合海藻酸钠水凝胶球3D培养软骨细胞抑制细胞去分化的研究 |
| The Application of Sodium Alginate Hydrogel Beads with Hyaluronic Acid in the Three-dimensional Culture of Chondrocytes to Inhibit the Dedifferentiation of Chondrocytes |
| Received:October 22, 2016 Revised:November 10, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.16.012 |
| 中文关键词: 软骨细胞 透明质酸钠 海藻酸钠 水凝胶 3D培养 |
| 英文关键词: Chondrocytes Sodium alginate Alginate Hydrogel Three-dimensional culture |
| 基金项目:国家自然科学基金项目（31170936, 31470936） |
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| 中文摘要: |
| 摘要 目的：验证海藻酸钠（Alginate, Alg）水凝胶球添加透明质酸钠（Sodium hyaluronate, HA）后，对大鼠膝软骨细胞体外3D培养时软骨细胞去分化的抑制情况。方法：用Ⅱ型胶原酶消化法获取2周龄SD大鼠膝关节原代软骨细胞，体外培养传代至P2。细胞分为2组，1组用2%海藻酸钠水凝胶球3D培养（Alg组），另一组用2%海藻酸钠+1%透明质酸钠的水凝胶球3D培养（Alg/HA组），采用正交法制作海藻酸钠水凝胶球。培养14天后，两组各取水凝胶球进行死活细胞染色观察细胞状态。其他水凝胶球收集后用4%多聚甲醛固定，30%蔗糖溶液脱水，OCT（Optimal cutting temperature compound）包埋剂包埋切片，采用苏木精 - 伊红染色法（Hematoxylin-eosin staining, HE染色）、甲苯胺蓝染色观察软骨细胞形态；采用阿利新蓝染色观察对比糖胺聚糖（Glycosaminoglycan, GAG）含量；采用免疫荧光染色对比Ⅱ型胶原（Collagen type Ⅱ, Col Ⅱ）含量；采用蛋白质免疫印迹法（Western blot）检测软骨细胞Col Ⅱ和聚蛋白多糖（Aggrecan, ACAN）的表达情况。结果：死活细胞染色显示两组水凝球内软骨细胞状态良好，几乎无死细胞；阿利新蓝染色显示Alg/HA组与Alg组相比软骨细胞分泌更多的GAG；免疫荧光染色显示Alg/HA组比Alg组软骨细胞内含更多的Ⅱ型胶原；Western blot显示Alg/HA组软骨细胞比Alg组Col Ⅱ的蛋白表达更多。结论：含透明质酸钠的海藻酸钠水凝胶可以更好地维持软骨细胞的表型，抑制软骨细胞去分化。 |
| 英文摘要: |
| ABSTRACT Objective: To verify the inhibitory effect of sodium alginate hydrogel beads with hyaluronic acid on the dedifferentiation of chondrocytes in vitro. Methods: Chondrocytes were obtained from knee articular of 2-week-old SD rat with type Ⅱ collagenase digestion method and cultured in vitro. P2 cells were divided into 2 groups. One was cultivated within 2% sodium alginate beads 3D culture (Alg group), another was cultivated within 2% sodium alginate + 1% sodium hyaluronate beads 3D culture (Alg/HA group). 14 days later, part of the beads were stained by live/dead cell staining, and others were harvested, fixed by 4% poly formaldehyde, OCT（Optimal cutting temperature compound） embedded and frozenly sectioned. Hematoxylin - eosin staining(HE staining) was used to observe the morphology of chondrocytes in the beads; Alcian blue staining was used for observation and comparison of glycosaminoglycan (GAG) content in chondrocytes. The immunofluorescence staining was used for comparison of type Ⅱ collagen (Col Ⅱ) content. Western blot was used to detect the expression of Col Ⅱ. Results: Live/ dead cell staining showed that there were few dead chondrocytes in 2 groups of alginate beads. Alcian blue staining showed that chondrocytes in Alg/HA group secreted more GAG than those in Alg group. Immunofluorescence showed that chondrocytes in Alg/HA group produced more Col Ⅱ than those in Alg group. Western blot showed that the Col Ⅱ expression of chondrocytes in Alg/HA group was higher than that in Alg groups. Conclusion: Alginate beads with sodium hyaluronate could maintain the chondrogenic phenotype and inhibit the dedifferentiation of chondrocytes better. |
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