PKH26标记和分子荧光活体成像技术在软骨组织工程研究中的应用  

Evaluation of Cartilage Engineering Using PKH26 and Molecular Light Imaging System

作　　者：祁霁舟 徐宝山[2] 彭江[3] 许文静[3] 杨强[2] 

机构地区：[1]天津医科大学研究生院,300070 [2]天津市天津医院脊柱外科 [3]中国人民解放军总医院

出　　处：《天津医药》2014年第12期1156-1159,I0010,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;2012T50235);天津市卫生局科技基金(2013KR16)

摘　　要：目的探讨PKH26荧光标记和分子荧光活体成像技术在软骨组织工程中的应用。方法用PKH26荧光标记犬软骨细胞,种植到多孔支架上,体外培养1周后异位移植到裸鼠背部,4周后用分子荧光活体成像系统示踪,并与X线检查结果对比。然后处死裸鼠取材,与免疫组织化学染色和免疫荧光观察结果对比。结果 4周分子荧光活体成像系统观察裸鼠背部标本处呈圆形强荧光,表明组织工程软骨在裸鼠体内生长良好。组织学切片结果显示番红O染色、Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性,荧光显微镜下观察结果显示组织工程化软骨中细胞均呈红色荧光,Ⅱ型胶原免疫荧光染色呈绿色荧光,叠加后呈黄色荧光。结论 PKH26荧光标记和分子荧光活体成像2种方法结合应用于软骨组织工程中,能够较理想地且大体无创伤性评估组织工程化软骨组织在体内的生长情况。Objective To investigate the application of PKH26 and molecular light imaging system in cartilage en-gineering. Methods Canine chondrocyte was labeled by fluorescent dye PKH26 and seeded into the porous cartilage acel-lular matrix scaffold. The cells/scaffold constructs were cultured in vitro for 1 week. Then the constructs were implanted intothe dorsal pocket of nude mice. We utilized a molecular light imaging system to macroscopically observe cells/scaffold con-structs in vivo with fluorescence at the 4thweeks, and compared with X-rays taken at the same position. The fluorescence im-ages were compared with the immunohistochemical and immunofluorescent results of cartilage-like tissue in vivo. Results Luminescent images were acquired at the 4thweeks, a red color enhanced overlay of the luminescent image over X-ray photo-graphic image demonstrated the location of the implants and the cell viability and cell growth on porous CACM scaffold in vivo were very well. Histological results show that the safranin O, anti-collagen Ⅱ immunohistochemistry and toluidine bluestain of cartilage- like tissue is positive. Immunofluorescence examination demonstrated chondrocytes in the constructswhitch is showen red fluorescence, and anti-collagenⅡ immunofluorescent staining was showen in green while the overlap-ping image is showen in yellow. Conclusion This study outlines an applicable non-destructive method to evaluate cellgrowth in tissue engineering constructs in vivo using PKH26 and molecular light imaging system.

关 键 词：软骨细胞 组织工程 荧光 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]