组织块法培养原代人PDLCs技术的新探讨

The new study of the tissue-explant technique on culturing primary human PDLCs

目的探讨组织块法培养原代人PDLCs生长情况和成功率,以及提高组织块法培养原代人PDLCs成功率的方法。方法采用组织块法培养原代人PDLCs。免疫组化ABC法进行波形丝蛋白(VIM)和细胞角蛋白(CK)的免疫组化染色,进行细胞来源鉴定。倒置显微镜下进行形态学观察,取第4代人PDLCs用MTT法绘制生长曲线。计算细胞培养成功率。结果组织块法培养原代人PDLCs成功率52.4%。波形丝蛋白染色阳性,细胞角蛋白染色阴性。细胞大多呈长梭形,胞体丰满,伸展良好,核仁清晰,细胞排列均匀,呈漩涡状,火焰状。生长曲线示细胞第3天开始进入对数生长期。结论本实验组织块法培养原代人PDLCs的成功率为52.4%,为人PDLCs作为种子细胞的牙周组织工程的研究,提供了实验基础和理论依据。

Objective To culture Primary human PDLCs by using tissue-explant technique, and to enhance the success rate of human PDLCs culture in vitro. Methods Human PDLCs were cultured using tissue-explant technique. The cell phenotype was identified by cytokeratin and Vimentin immunohistoehemieal staining. Cellular morphology and cell growth were observed and photographed under inverted microscope. The fourth passage human PDLCs were taken to measure cells growth curve by MTF assay. Results The success rate of human PDLCs culture in vitro was increased to 52.4%. In the immunohistochemical study, vimentin staining was positive and cytokeratin staining was negative. Human PDLCs were long shuttle shaped in vitro, bodies of cells were plump,cells spreaded well and nucleolus was clear. Cells arranged in a certain orientation, looked like flame or swirl. Cells growth curve showed that human PDLCs entered logarithm proliferative stage from the third day after passage. Conclusion Human PDLCs were successfully cultured with tissue-explant technique and the success rate of human PDLCs culture in vitro was increased.