中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (14): 2161-2165.doi: 10.3969/j.issn.2095-4344.3092

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

体外培养SD大鼠关节软骨细胞原代至第3代的形态学特点

杨  帆1，2，刘保一2，刘家河2，杨佳慧2，覃开蓉1，赵德伟1，2    

  1. 1大连理工大学生物医学工程学院，辽宁省大连市   116024；2大连大学附属中山医院骨科，辽宁省大连市   116001
  • 收稿日期:2020-04-01 修回日期:2020-04-09 接受日期:2020-05-16 出版日期:2021-05-18 发布日期:2020-12-30
  • 通讯作者: 赵德伟，博士，主任医师，教授，大连理工大学生物医学工程系，辽宁省大连市 116024；大连大学附属中山医院骨科，辽宁省大连市 116001
  • 作者简介:杨帆，男，1987年生，黑龙江省大庆市人，汉族，大连理工大学在读博士，医师，主要从事生物材料和骨科学相关的实验研究。
  • 基金资助:
    国家自然科学基金委项目(81672139，81371942)，项目负责人：赵德伟；国家重点研发计划(2016YFC1102000)，项目负责人：赵德伟；大连市科技创新基金项目(2018J11CY030)，项目负责人：赵德伟

Morphological characteristics of in vitro cultured articular chondrocytes from Sprague-Dawley rats at passage number from 0 to 3

Yang Fan1, 2, Liu Baoyi2, Liu Jiahe2, Yang Jiahui2, Qin Kairong1, Zhao Dewei1, 2   

  1. 1School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; 2Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • Received:2020-04-01 Revised:2020-04-09 Accepted:2020-05-16 Online:2021-05-18 Published:2020-12-30
  • Contact: Zhao Dewei, MD, Chief physician, Professor, School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • About author:Yang Fan, MD candidate, Physician, School of Biomedical Engineering, Dalian University of Technology, Dalian 116024, Liaoning Province, China; Department of Orthopedics, Zhongshan Hospital, Dalian University, Dalian 116001, Liaoning Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81672139 and 81371942 (both to ZDW); the National Key Research & Development Plan of China, No. 2016YFC1102000 (to ZDW); the Scientific Innovation Foundation of Dalian, No. 2018J11CY030 (to ZDW)

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摘要：

文题释义：
胶原酶(collagenase)：是从溶组织梭状细胞芽孢杆菌提取制备的，主要水解结缔组织中胶原蛋白成分。当拟消化的组织较硬，内含较多结缔组织或胶原成分时，用胰蛋白酶解离细胞的效果较差，这时可采用胶原酶解离细胞法。 胶原酶仅对细胞间质有消化作用而对上皮细胞影响不大。胶原酶分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ型以及肝细胞专用胶原酶，要根据所要分离消化的组织类型选择胶原酶类型。
软骨细胞：关节软骨组织中的唯一细胞成分为软骨细胞，软骨细胞主要起到维持关节软骨组织内环境稳态的作用。软骨细胞位于软骨基质内的软骨陷窝中，在其周围有一层由胶原及蛋白聚糖等大分子构成的染色深的软骨囊，构成细胞外基质的重要成分为胶原，其中II型胶原约占90%-95%。关节软骨组织损伤后自我修复能力极差的原因是其组织内无神经、血管，软骨细胞增值能力有限。
细胞培养：是指在生物体外模拟体内环境(无菌、温度、酸碱度和营养条件等)，使分离出的细胞在其中生存、生长、繁殖并维持其主要结构和功能的技术。

背景：常用的获得软骨细胞的方法有机械-酶消化法、链霉蛋白酶和胶原酶序贯消化法、胰蛋白酶和Ⅱ型胶原蛋白酶序贯消化法以及单纯Ⅱ型胶原蛋白酶消化法。
目的：探讨SD大鼠膝关节软骨细胞Ⅱ型胶原酶体外分离、提取、鉴定方法，观察原代至第3代软骨细胞的形态学特点。
方法：课题组采用一步酶消化法(Ⅱ型胶原酶)体外分离、提取、培养SD大鼠膝关节软骨细胞并进行传代培养，进而构建其体外培养体系。使用细胞增殖实验、倒置相差显微镜观察细胞组织学形态；甲苯胺蓝染色、Ⅱ型胶原与蛋白聚糖免疫荧光染色等方法进行细胞性状鉴定。实验经大连大学附属中山医院伦理委员会批准。
结果与结论：①镜下观察与细胞增殖实验均显示第2，3代细胞形态、增殖能力最佳，后逐渐退化；②甲苯胺蓝染色、Ⅱ型胶原与蛋白聚糖免疫荧光染色等组织学检测显示，第3代体外培养软骨细胞状态功能良好；③结果表明，一步酶消化法(Ⅱ型胶原酶)能够成功地从SD大鼠膝关节标本中分离出软骨细胞并且可以进行传代培养；体外培养3代后软骨细胞去分化特性逐渐增强，第3代体外培养软骨细胞性状良好，可用于实验研究。

关键词: 软骨细胞, 酶消化法, 细胞培养, 大鼠, 实验

Abstract: BACKGROUND: Common methods for obtaining chondrocytes include mechanical-enzymatic digestion, sequential digestion of pronase and collagenase, sequential digestion of trypsin and type II collagenase, and simple type II collagenase digestion.
OBJECTIVE: To investigate the isolation, extraction and identification of knee joint chondrocytes from Sprague-Dawley rats, and to observe the morphological characteristics of chondrocytes from primary to passages 3.
METHODS: We used single-step digestive approach (type II collagenase digestion) to isolate and obtain chondrocytes from the knee joints of Sprague-Dawley rats in vitro, and then established a culture system in vitro. Cell proliferation assay and inverted phase contrast microscope were used to observe morphohistology of the cells. Toluidine blue staining, type II collagen as well as proteoglycan immunofluorescence staining were used to identify the characteristics of chondrocytes. The experimental protocol was approved by the Ethics Committee of Zhongshan Hospital of Dalian University.
RESULTS AND CONCLUSION: Microscopic observation and cell proliferation assay both showed satisfactory cell morphology and proliferation at passages 2-3, and the capacity of the cells decreased gradually. Moreover, toluidine blue staining, type II collagen and proteoglycan immunofluorescence staining revealed superior characteristics of passage 3 chondrocytes cultured in vitro. These findings indicate that chondrocytes can successfully be isolated by the single-step type II collagenase digestion. Cell proliferation and passage cultivation can also be achieved. And dedifferentiation capacity of chondrocytes is improved gradually after the passages 3 cultured in vitro. Passage 3 cells cultured in vitro have superior characteristics for the use in experimental researches.

Key words: chondrocyte, enzyme digestion, cell culture, rat, experiment

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