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目的:探讨如何以简便快捷的方法获得大量最佳生长状态的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞。方法:用2.5g/L胰酶、35g/L热溶素和9.0×103U/LDispase分别在A,B,C不同条件下处理皮肤标本,观察表皮与真皮的分离情况,并分别培养角朊细胞和真皮成纤维细胞,观察比较细胞的贴壁率和克隆形成率,通过MTT和BrdU检测观察细胞的生长状态,并分别用不同方法获得的细胞构建组织工程皮肤。结果:A,B,C3种方法角朊细胞的贴壁率(%)分别为:8.7±0.2,12.1±0.2,15.9±0.4;成纤维细胞的贴壁率(%)分别为:20.2±2.4,30.5±1.8,38.3±1.9。组间比较差异有统计学意义(t=3.3506,t0.05/2,18=2.101,P<0.05)。角朊细胞克隆形成率(%)分别为:0.110±0.025,0.260±0.026,0.370±0.021。组间比较差异有统计学意义(t=2.2875,t0.05/2,18=2.101,P<0.05)。在各种条件下,Dispase处理组获得的细胞数量、细胞贴壁率和克隆形成率均优于其他消化液处理组,细胞纯度高,生长状态良好;用其他方法获得的细胞生长状态相似,都可成功构建组织工程皮肤。结论:使用9.0×103U/LDispase可以简便快捷的获得大量生长状态良好的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞,合适的消化方法、时间和温度可以最大限度保持原代细胞的活力
Objective: To explore how to obtain a large number of keratinocytes and dermal fibroblasts in optimal growth state as seed cells for skin tissue engineering in a simple and quick way. Methods: The skin specimens were treated with 2.5g / L trypsin, 35g / L hot-melt and 9.0 × 103U / LDispase under the different conditions of A, B and C to observe the separation of epidermis and dermis, and to separately culture keratinocytes And dermal fibroblasts were observed. The adherent rate and clonogenic rate of cells were observed. MTT and BrdU detection were used to observe the growth of cells. Tissue-engineered skin was constructed by using different methods. Results: The attachment rates (%) of keratinocytes in A, B and C3 methods were 8.7 ± 0.2, 12.1 ± 0.2 and 15.9 ± 0.4, respectively. The adherent rates of fibroblasts were 20.2 ± 2.4 , 30.5 ± 1.8, 38.3 ± 1.9. The difference between the two groups was statistically significant (t = 3.3506, t0.05 / 2,18 = 2.101, P <0.05). The rate of keratinocyte colony formation (%) was respectively 0.110 ± 0.025, 0.260 ± 0.026 and 0.370 ± 0.021. The difference between the two groups was statistically significant (t = 2.2875, t0.05 / 2,18 = 2.101, P <0.05). Under the various conditions, the number of cells, cell attachment rate and colony formation rate of Dispase treatment group were better than other digestive juice treatment group, the cell purity was high and the growth state was good; the cell growth state obtained by other methods was similar Tissue engineered skin can be successfully constructed. Conclusion: Using 9.0 × 103U / LDispase can quickly and easily obtain a large number of well-growing keratinocytes and dermal fibroblasts as seed cells for skin tissue engineering. Suitable digestion methods, time and temperature can keep the primary cells Vitality