CAJ | 학술논문

目的：探讨PKH26荧光标记在牛尾髓核细胞的应用，为髓核组织工程的体内研究提供可示踪的种子细胞。方法从牛尾椎间盘中分离髓核组织，通过酶消化法获取原代细胞，倒置显微镜下观察，P1代髓核细胞进行番红O、甲苯胺蓝和Ⅱ型胶原免疫细胞化学染色，对P1代髓核细胞进行PKH26荧光染料标记，并对标记后细胞的活性、荧光强度（0、14、28 d）、增殖特性及基因表达进行评估。结果分离得到的髓核细胞数为（1.56±0.35）×106/g，倒置显微镜下，原代细胞培养4 d开始贴壁，细胞成群生长，14 d铺满瓶底，原代细胞、P1代细胞均呈类软骨样细胞形态；P1代髓核细胞甲苯胺蓝染色异染、番红O染色和Ⅱ型胶原免疫细胞化学染色阳性；PKH26标记前后细胞活性均在95%以上，1、14、28 d呈递减趋势，但28 d时细胞仍可检测出较强的荧光，标记后的细胞增殖及基因表达（Ⅰ、Ⅱ型胶原及聚集蛋白聚糖）与标记前的细胞相比差异无统计学意义（P＞0.05）。结论牛尾髓核组织中可以分离出数量满意的髓核细胞，且具有软骨样细胞的表型，可以用作种子细胞；PKH26标记后的髓核细胞无生物学特性的变化，可以用作髓核细胞体内研究的示踪染料。
목적：탐토PKH26형광표기재우미수핵세포적응용，위수핵조직공정적체내연구제공가시종적충자세포。방법종우미추간반중분리수핵조직，통과매소화법획취원대세포，도치현미경하관찰，P1대수핵세포진행번홍O、갑분알람화Ⅱ형효원면역세포화학염색，대P1대수핵세포진행PKH26형광염료표기，병대표기후세포적활성、형광강도（0、14、28 d）、증식특성급기인표체진행평고。결과분리득도적수핵세포수위（1.56±0.35）×106/g，도치현미경하，원대세포배양4 d개시첩벽，세포성군생장，14 d포만병저，원대세포、P1대세포균정류연골양세포형태；P1대수핵세포갑분알람염색이염、번홍O염색화Ⅱ형효원면역세포화학염색양성；PKH26표기전후세포활성균재95%이상，1、14、28 d정체감추세，단28 d시세포잉가검측출교강적형광，표기후적세포증식급기인표체（Ⅰ、Ⅱ형효원급취집단백취당）여표기전적세포상비차이무통계학의의（P＞0.05）。결론우미수핵조직중가이분리출수량만의적수핵세포，차구유연골양세포적표형，가이용작충자세포；PKH26표기후적수핵세포무생물학특성적변화，가이용작수핵세포체내연구적시종염료。
Objective To investigate the application of PKH26 fluorescent labeling on nucleus pulposus cells isolat-ed from bovine coccyx disc, and to provide nucleus pulposus tissue engineering with traceable nucleus pulposus cells by PKH26 fluorescence labelling. Methods Nucleus pulposus primary cells were isolated from the nucleus pulposus tissue de-tached from bovine coccyx disc by enzymatic digestion, and observed under the inverted microscope. Safranin O, toluidine blue and type Ⅱ collagen immunocytochemistry methods used to stain for passage one generation cells. Nucleus pulposus cells were labeled with PKH26 fluorescence in accordance with the instructions. The cell activity, fluorescence intensity at d0, d14 and d28 of culture, characteristics of proliferation and the expression of gene in labeled cells were assessed. Re-sults Isolated nucleus pulposus cells amounted to (1.56 ± 0.35) × 106/g. Under the inverted microscope, primary cells ad-hered at the 4 th day of culture, grew in groups, and covered the bottom of culture flask at the 13 th day. Both primary cells and the P1 generation cells were chondrocyte-like morphology. The staining of safranin O, toluidine blue and typeⅡcolla-gen immunocytochemistry for P1 generation of nucleus pulposus cells showed positive results. The cell activity before and af-ter PKH26 labeling showed more than 95%, and the fluorescence intensity at d0, d14 and d28 performed a decreasing trend, but still showed detect strong fluorescence at d28. There were no significant differences in proliferation and the expression of gene (collagen typeⅠandⅡ, aggrecan) before and after cell labeling (P>0.05). Conclusion As the seed cells of tissue en-gineering, nucleus pulposus cells isolated from bovine coccyx can reach a satisfactory number and maintain cartilage-like phenotype, and no changes shown in the biological characteristics after labeling. PKH26 labeled nucleus pulposus cells are suitable for the traceable cells in vivo study.