纤维蛋白凝胶移植培养口腔黏膜细胞的研究

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1×106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(4.62×103个/cm2),组织有多量血管形成(5.72×103个/cm2),对照组中上皮管腔(1.76×103 个/cm2)及血管数量少(0.88×103 个/cm2),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11 d,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18.5 d,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.

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

Objective To investigate an optimal delivery vehicle for cultured keratinocyte transplantation.Methods Human oral epithelium keratynocytes were cultured 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 1×106/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),4.62×103/cm2 and 5.72×103/cm2 respectively,were found in the subcutaneous fascia 3 weeks after transplantation,and the difference compared with the DMEM:F12 group(1.76×103/cm2 and 0.88×103/cm2)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 cultured oral keratinocyte into an excellent functional epithelium tissue.So it can be used as good delivery vehicle for cultured keratinocyte transplantation.