Flow cytometric analysis of the human articular chondrocyte phenotype in vitro.
Flow cytometric analysis of the human articular chondrocyte phenotype in vitro.
复制标题
体外人关节软骨细胞表型的流式细胞术分析。
作者：
L. Wang;G. Verbruggen;K. Almqvist;D. Elewaut;C. Broddelez;E. Veys
目的
建立流式细胞术检测人关节软骨细胞表型的方法，并对不同体外培养体系中的软骨细胞进行验证。
方法
通过在不同体外条件下培养细胞来调节软骨细胞表型：即在单层中和在凝胶琼脂糖中悬浮培养。单层培养的软骨细胞表型进行了测定与软骨细胞特异性aggrecan，II型和I型胶原的单克隆抗体免疫组化染色。流式细胞术被用来量化软骨细胞表达这些细胞外基质分子在两种培养条件下的比例。为了排除细胞收获方法对细胞结合的ECM分子的存在的影响，使用非蛋白水解分离程序获得软骨细胞用于流式细胞术。用EDTA分离来自单层培养物的亚融合细胞。琼脂糖经琼脂酶消化后，获得琼脂糖凝胶中培养的软骨细胞。
结果
免疫组化染色显示，单层培养的软骨细胞，在血清的存在下，逐渐失去软骨细胞特异性聚集蛋白聚糖和II型胶原的表达，而I型胶原表达增加。流式细胞术允许单层培养的软骨细胞表型进行评估可重复。软骨细胞表型通过细胞膜相关的细胞外基质抗原来表征。EDTA，用于从单层培养物中获得单细胞，不影响细胞相关基质。当软骨细胞在凝胶琼脂糖中培养时，流式细胞术可以定量软骨细胞保持或重新表达其原始表型的百分比。用于从琼脂糖凝胶中分离细胞的琼脂酶消化程序不影响质膜相关的细胞外基质抗原。
结论
流式细胞术允许定量表达聚集蛋白聚糖、II型和I型胶原蛋白的细胞在其细胞相关的细胞外基质中。特异性单克隆抗体的不断增加将拓宽该方法的应用范围。
OBJECTIVE
To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems.
METHODS
Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase.
RESULTS
Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens.
CONCLUSION
Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.
影响因子：
2.4
作者：
J. Kumagai;K. Sarkar;H. Uhthoff;Y. Okawara;A. Ooshima
通讯作者：
J. Kumagai;K. Sarkar;H. Uhthoff;Y. Okawara;A. Ooshima
影响因子：
15.9
作者：
GOLDRING, MB;BIRKHEAD, JR;APPERLEY, JF
通讯作者：
APPERLEY, JF
影响因子：
4
作者：
Hans J. Häuselmann;Russell J. Fernandes;Su S. Mok;Thomas M. Schmid;Joel A. Block;M. Aydelotte;Klaus E. Kuettner;E. Thonar
通讯作者：
Hans J. Häuselmann;Russell J. Fernandes;Su S. Mok;Thomas M. Schmid;Joel A. Block;M. Aydelotte;Klaus E. Kuettner;E. Thonar