中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (32): 5097-5101.doi: 10.12307/2022.909

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

老年小鼠踝关节软骨细胞体外分离及培养鉴定

贾彩霞1，2，3，何  斯2，3，4，哈小琴2，3   

  1. 1兰州大学公共卫生学院，甘肃省兰州市  730000；2中国人民解放军联勤保障部队第九四〇医院，甘肃省兰州市  730050；3甘肃省干细胞与基因药物重点实验室，甘肃省兰州市  730050；4兰州大学第二临床医学院，甘肃省兰州市  730000
  • 收稿日期:2021-10-20 接受日期:2021-12-10 出版日期:2022-11-18 发布日期:2022-05-12
  • 通讯作者: 哈小琴，博士， 教授，主任医师，中国人民解放军联勤保障部队第九四〇医院，甘肃省兰州市 730050； 甘肃省干细胞与基因药物重点实验室，甘肃省兰州市 730050
  • 作者简介:贾彩霞，女，1996年生，甘肃省白银市人，汉族，兰州大学在读硕士，主要从事干细胞修复损伤组织器官基础与应用研究。
  • 基金资助:
    甘肃省拔尖领军人才培养(2020808)，项目负责人：哈小琴；中央高校基本科研业务费项目(31920200020)，项目负责人：哈小琴；兰州市人才培养创新创业项目(2016-RC-61)，项目负责人：哈小琴

In vitro isolation, culture and identification of chondrocytes from the ankle joint of aged mice

Jia Caixia1, 2, 3, He Si2, 3, 4, Ha Xiaoqin2, 3   

  1. 1School of Public Health, Lanzhou University, Lanzhou 730000, Gansu Province, China; 2The 940th Hospital of Joint Logistics Support Force of PLA, Lanzhou 730050, Gansu Province, China; 3Key Laboratory of Stem Cell and Gene Medicine of Gansu Province, Lanzhou 730050, Gansu Province, China; 4The Second Clinical Medical College of Lanzhou University, Lanzhou 730000, Gansu Province, China
  • Received:2021-10-20 Accepted:2021-12-10 Online:2022-11-18 Published:2022-05-12
  • Contact: Ha Xiaoqin, MD, Professor, Chief physician, The 940th Hospital of Joint Logistics Support Force of PLA, Lanzhou 730050, Gansu Province, China; Key Laboratory of Stem Cell and Gene Medicine of Gansu Province, Lanzhou 730050, Gansu Province, China
  • About author:Jia Caixia, Master candidate, School of Public Health Lanzhou University, Lanzhou 730000, Gansu Province, China; The 940th Hospital of Joint Logistics Support Force of PLA, Lanzhou 730050, Gansu Province, China; Key Laboratory of Stem Cell and Gene Medicine of Gansu Province, Lanzhou 730050, Gansu Province, China
  • Supported by:
    the Top-notch and Leading Talents in Gansu Province, No. 2020808 (to HXQ); the Fundamental Research Funds for the Central Universities, No. 31920200020 (to HXQ); Lanzhou Municipal Talent Training Innovation and Entrepreneurship Project, No. 2016-RC-61 (to HXQ)

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摘要：

文题释义：
软骨细胞：位于软骨基质内的腔隙，即软骨陷窝，包括幼稚软骨细胞和成熟软骨细胞，幼稚软骨细胞单个分布于软骨周边，细胞小，扁圆形，随着细胞逐渐长大，会呈现圆或椭圆形，核小，即成熟软骨细胞。2-8个成熟软骨细胞会聚集成群，位于软骨中央，称为同源细胞群，是由一个幼稚软骨细胞分裂而成。
贴壁细胞：这类细胞的生长必须有可以贴附的支持物表面，并且依靠自身分泌的或培养基中添加的贴附因子才能生长繁殖，其贴附后的形态一般为成纤维细胞样或上皮细胞样。它的生长过程一般包括游离期、贴壁期、潜伏期、对数期、平台期和衰退期。

背景：老年小鼠的关节软骨本身存在一定的磨损，提取难度较大；尤其是踝关节等难以分离关节软骨面的小关节软骨细胞的提取方法更加少见。
目的：探究实验室改良两步酶消化法提取老年小鼠踝关节软骨细胞的效果，体外培养观察并评价其生物学特性。 
方法：选取46周龄老年BALB/c雄性小鼠踝关节，通过实验室改良的0.25%胰酶EDTA、2 g/L Ⅱ型胶原酶两步酶消化法顺序进行分离提取及体外培养。使用倒置显微镜观察细胞生长形态，锥虫蓝染色并计数原代贴壁细胞，甲苯胺蓝和Ⅱ型胶原免疫荧光染色，二甲基亚甲基蓝显色法测定原代贴壁细胞培养液和消化液中硫酸糖胺多糖水平，CCK-8试剂盒检测软骨细胞增殖进行软骨细胞生物学特性鉴定及评价。
结果与结论：①关节软骨经两步酶消化后，细胞全部解离释放，老年小鼠每个踝关节平均收获9×105个原代贴壁细胞；原代及第1代、第2代细胞贴壁形态为梭形和多角形；②细胞甲苯胺蓝染色可见蓝紫色易染颗粒，Ⅱ型胶原免疫荧光染色可见红色荧光阳性表达；③原代细胞培养液和消化液中硫酸糖胺多糖分别为(42.41±15.00)，(65.63±10.00) mg/L；阴性对照中硫酸糖胺多糖分别为(0.35±3.78)，(0.21±2.56) mg/L；④原代细胞第6天细胞数量达到最大，第1代和第2代细胞第5天达到最大，同一时间点，第1代细胞的细胞活力大于第2代细胞和原代细胞；⑤结果证实：实验室改良的两步酶消化法可成功分离老年小鼠踝关节软骨细胞，贴壁细胞纯度高、数量多，可成功合成和分泌Ⅱ型胶原及蛋白聚糖，具有良好的生物学特性，其中第1代软骨细胞的活力最高，第2代次之，可用于实验研究。      

https://orcid.org/0000-0002-9337-7581 (贾彩霞) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程  

关键词: 老年小鼠, 踝关节, 软骨细胞, 酶消化法, 细胞培养

Abstract: BACKGROUND: Knee cartilage loss certainly occurs in aged mice, so it is difficult to extract the articular cartilage from the mice. In particular, it is difficult and rarely reported to extract chondrocytes from the small joint on the articular cartilage surface of the ankle joint.  
OBJECTIVE: To explore the effect of an improved two-step enzyme digestion method to extract chondrocytes from the ankle joint of aged mice, and to observe and evaluate their biological characteristics in vitro. 
METHODS: The ankle joints of 46-week-old mice were selected, separated and extracted sequentially by the improved two-step enzymatic digestion method in the laboratory with 0.25% pancreatin EDTA and 2g/L type II collagenase. Extracted cells were cultured in vitro and cell growth morphology was observed with an inverted microscope. After trypan blue staining, the number of primary adherent cells was calculated. Toluidine blue and type II collagen immunofluorescence staining was conducted. Chromogenic method using dimethyl methylene blue was used to determine the content of glycosaminoglycan sulfate in the primary adherent cell culture and digestive fluid. Proliferation of chondrocytes was detected using cell counting kit-8 to identify and evaluate the biological characteristics of chondrocytes.
RESULTS AND CONCLUSION: After the two-step enzymatic digestion of the articular cartilage, all cells were dissociated and released. An average of 9×105 primary adherent cells were harvested from each ankle joint of aged mice. Primary cells and the first- and second-generation cells displayed spindle and polygonal morphology. Toluidine blue staining showed blue-purple particles that were easily dyed. Type II collagen immunofluorescence staining revealed the positive expression of red fluorescence. The contents of glycosaminoglycan sulfate in the primary adherent cell culture fluid and digestive fluid were (42.41±15.00) and (65.63±10.00) mg/L, which were (0.35±3.78) and (0.21±2.56) mg/L in the negative control culture fluid and digestive fluid, respectively. The number of primary cells reached the maximum on the 6th day, and the number of the first- and second-generation cells reached the maximum on the 5th day. Meanwhile, the cell viability of the first-generation cells was greater than that of the second-generation cells and the primary cells. To conclude, the improved two-step enzymatic digestion method can successfully isolate chondrocytes from the ankle joint of aged mice. Large amounts of adherent cells with high purity and good biological characteristics can successfully synthesize and secrete type II collagen and proteoglycan. The first-generation chondrocytes have the highest viability, followed by the second-generation cells, which can be used for experimental research.

Key words: aged mice, ankle joint, chondrocyte, enzyme digestion method, cell culture

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