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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。
Objective To investigate the application of PKH26 fluorescent labeling in cow’s nucleus pulposus cells and to provide traceable seed cells for in vivo study of nucleus pulposus engineering. Methods The nucleus pulposus was isolated from the caudal disc and the primary cells were obtained by enzyme digestion. The cells were observed under inverted microscope. The nucleus pulposus cells of P1 were subjected to immunocytochemical staining with Safranin O, toluidine blue and collagen Ⅱ. The nuclear cells were labeled with PKH26 fluorescent dye, and the activity of the labeled cells, fluorescence intensity (0,14,28 d), proliferation characteristics and gene expression were evaluated. Results The number of nucleus pulposus cells was (1.56 ± 0.35) × 106 / g. Under inverted microscope, the primary cells cultured for 4 days started to adhere and the cells grew in groups. The primary cells, P1 The generation of cells showed cartilage-like morphology. The staining of toluidine blue staining, safranin O staining and typeⅡcollagen immunocytochemistry in nucleus pulposus P1 were all positive. The cell viability was above 95% before and after PKH26 labeling. , And showed a decreasing trend on the 28th day. However, the cells could still detect strong fluorescence on the 28th day. The marked cell proliferation and gene expression (type Ⅰ, type Ⅱ collagen and aggrecan) Statistical significance (P> 0.05). Conclusion The number of nucleus pulposus cells can be isolated from the nucleus pulposus and the phenotype of cartilage-like cells can be used as seed cells. PKH26-labeled nucleus pulposus cells have no biological changes and can be used as nucleus pulposus Cell In Vivo Study of Tracer Dyes.