PKH26荧光标记牛尾髓核细胞的实验研究  

An Experimental Study on Bovine Nucleus Pulposus Cells Labelled with PKH26 in Vitro

作　　者：丁晓明 徐宝山[2] 伍耀宏 许海委[2] 杨强[2] 马信龙[2] 张春秋[3] 李秀兰[2] 张扬[2] 

机构地区：[1]医科大学研究生院,天津300070 [2]天津市天津医院 [3]天津理工大学

出　　处：《天津医药》2014年第9期849-852,I0001,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81272046,31300798,31000432);中国博士后科学基金项目(2011M500530,2012T50221);天津市卫生局科技基金(2013KR16)

摘　　要：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 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 ＆#177; 0.35） ＆#215; 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＆gt;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.

关 键 词：组织工程 椎间盘 牛尾 髓核细胞 PKH26 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R349.89[医药卫生—基础医学]