纤维蛋白凝胶移植培养口腔黏膜细胞的研究  

Mucosa epithelium tube formation with cultured oral keratinocyte using fibrin glue as delivery vehicle

作　　者：刘立强[1] 焦安贵 李森恺[1] 范金财[1] 李养群[1] 刘元波[1] 

机构地区：[1]中国医学科学院北京协和医学院整形外科医院,北京100041 [2]山东省日照市碑廓医院

出　　处：《中华实验外科杂志》2008年第4期440-442,I0001,I0002,共5页Chinese Journal of Experimental Surgery

基　　金：卫生部临床学科重点资助项目（20010409）

摘　　要：目的为培养上皮细胞寻找良好的移植载体。方法用含自体血清培养基培养人口腔黏膜角质细胞，实验组细胞置于纤维蛋白凝胶中，对照组细胞置于DMEM：F12细胞培养液中，将悬液细胞以1×10^6个／ml浓度分别移植于裸鼠皮下和创面，于移植后1、2、3、4、6周取材，分别行苏木素-伊红染色及冰冻切片抗人HLA—Ⅰ免疫荧光染色及抗人Ⅳ型胶原及抗人层黏蛋白免疫组织化学检查。结果皮下移植时，凝胶组角质细胞逐步增殖分化形成较大上皮管腔（4．62×10^3个／cm^2），组织有多量血管形成（5．72×10^3个／cm^2），对照组中上皮管腔（1．76×10^3个／cm^2）及血管数量少（0．88×10^3个／cm^2）、，两者差异有统计学意义（P〈0．01），管腔上皮抗人-HLA免疫荧光阳性；实验组创面愈合平均时间为11d，愈合处皮肤粗糙、角化，抗人-HLA免疫荧光强阳性，免疫组织化学染色示基底膜形成完整，对照组愈合时间为18．5d，愈合创面凹陷、光滑无角化，抗人-HLA免疫荧光弱阳性，两组创面愈合时间差异有统计学意义（P〈0．05）。结论纤维蛋白凝胶可促进移植黏膜上皮细胞的增殖分化并形成功能完备的上皮组织，是上皮细胞移植的良好载体。Objective To investigate an optimal delivery vehicle for cultured keratinocyte transplantation. Methods Human oral epithelium keratynocytes were cuhured in medium containing autologous serum. Two groups were divided according to the delivery vehicle,fibrin gel or DMEM：F12. The cell suspensions were transplanted into subcutaneously or on the wound of nude mice respectively at a concentration of 1 × 10^6/ml. The specimens were taken at 1st ,2nd,3rd,4th and 6th week posttransplantation, and processed for immunofluorescence anti-HLA staining to determine the graft acceptance, and anti-human Ⅳ collagen and anti-human laminin immunohistochmical staining procedures to indicate the basement membrane formation. Results Rich blood vessels and lots of tubes lumen lined with viable epithelium （ positive for anti-HLA） ,4.62 × 10^3/cm^2 and 5.72 × 10^3/cm^2 respectively,were found in the subcutaneous fascia 3 weeks after transplantation,and the difference compared with the DMEM：F12 group （1.76 × 10^3/cm^2 and 0.88 × 10^3/cm^2 ） was significant. There was significant difference in average wound healing time between two groups （ 11 days in fibrin gel group and 18.5 days in DMEM：F12 group）. Collagen type Ⅳ and laminin were detected at the dermo-epidermal junction in fibrin gel group from day 14 postgraft, but not found in DMEM：F12 group. Conclusion Fibrin gel could promote the proliferation and differentiation of the cuhured oral keratinocyte into an excellent functional epithelium tissue. So it can be used as good delivery vehicle for cuhured keratinocyte transplantation.

关 键 词：移植 角质细胞 纤维蛋白凝胶 Ⅳ型胶原 层黏蛋白 

分 类 号：R78[医药卫生—口腔医学]