人牙龈成纤维细胞原代培养方法的比较研究

目的：建立和评价人牙龈成纤维细胞原代培养方法,并观察其生物学特性。方法：分别用组织块法、2种改良酶消组织块法培养人牙龈成纤维细胞（HGF）,用形态学、免疫荧光鉴定细胞来源。通过活细胞观察,MTT比色实验研究细胞体外生物特性。比较3种培养方法培养HGF的效果。结果：细胞抗波形丝蛋白染色阳性,抗角蛋白染色阴性,符合人牙龈成纤维细胞的形态学特征和生物学特性。组织块法、改良酶消组织块法（翻瓶法）、改良酶消组织块法（盖玻片法）的细胞培养成功率分别为26.7%、54%、60%。组织块法和2种改良酶消组织块法间的成功率差异有显著性（P〈0.01）,2种改良酶消组织块法间的成功率差异无显著性（P〉0.05）。结论：本实验建立的细胞系为人牙龈成纤维细胞。2种改良酶消化组织块法可显著提高人牙龈成纤维细胞原代培养成功率。

Comparation of primary culture methods in vitro of human gingival fibroblast

Objective: To establish and evaluate the primary culture methods of human gingival fibroblast (HGF) cell lines and to investigate their biological features. Methods: The primary Cells were cultured with tissue explants method ,and two improved enzymatic digestion-explants methods respectively. The Cells were identified by means of morphological analysis and immunofluorescence. The biological features was investigated with inverted phase contrast microscope and MTT colorimetric assay. Compare the effectiveness of the three methods in primary culture of HGF. Results: Cultured cells were spindle-shaped,and had a positive reaction to antibodies against vimentin and a negative reaction to antibodies against keratin. Their morphological and biological characteristics were similar to those of typical HGF. The success rate of primary cells culture were 26.7%(tissue explants method),54%(improved enzymatic digestion -explants method invert flask ),60%(improved enzymatic digestion -explants method coverslip ) respectively. There was statistically significant difference between tissue explants method and the two enzymatic digestion-explants methods(P <0.01). There was no statistically significant difference between the two enzymatic digestion-explants methods(P >0.05). Conclusion: The cell lines established in this study are proved to be human gingival fibroblasts. The two enzymatic digestion-explants methods can noticeably increase the success rate of primary culture of human gingival fibroblast.