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

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 typeⅡ,ColⅡ)含量;采用蛋白质免疫印迹法(Western blot)检测软骨细胞ColⅡ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ColⅡ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。

Objective： To verify the inhibitory effect of sodium alginate hydrogel beads with hyaluronic acid on the dedifferentiation of chondrocytes in vitro. Methods： Chondroeytes were obtained fi：om 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（liE staining） was used to observe the morphology ofchondrocytes in the beads; Aleian 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 AIg/HA group secreted more GAG than those in Alg group. Immunofluorescence showed that chondrocytes in AIg/HA group produced more ColⅡ 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. Conclusions： Alginate beads with sodium hyaluronate could maintain the chondrogenic phenotype and inhibit the dedifferentiation of chondrocytes better.