人牙周膜干细胞的生物学活性研究

Study on Biological Activity of Human Periodontal Ligament Stem Cells

目的体外分离培养、鉴定人牙周膜干细胞（PDLSCs）并探讨其生物学活性的研究.方法应用酶解组织块法分离培养PDLSCs,应用相差显微镜观察细胞表型变化;HE染色及免疫组化染色进行形态学检测;流式细胞术检测细胞表面CD29,CD44,CD105,CD34,CD45和HLA-DR的表达;诱导成骨细胞、成脂肪细胞及成神经元分化,并进行特异性染色分析,扫描电镜观察,检测PDLSCs生物学活性.结果 体外成功分离培养人PDLSCs,HE染色显示细胞均一性好;角蛋白阴性,波形蛋白和CD44阳性表达;流式细胞仪结果显示PDLSCs表面高表达CD29（92.47%）,CD44（96.42%）和CD105（96.40%）;CD34,CD45和HLA-DR阴性表达;诱导成骨结果显示PDLSCs有较强的成骨活性,茜素红染色诱导组有明显的矿化结节形成,ALP染色阳性;成脂诱导PDLSCs有较强的成脂活性,油红＂O＂脂肪染色阳性;成神经元诱导后NSE免疫组化染色呈阳性.结论 PDLSCs在体外具有多向分化的潜能.

Objective To isolate and culture in vitro, the identification of human periodontal ligament stem cells （PDLSCs） and to explore its biological activity. Methods Application of enzymatic tissue explant isolated and cultured in vitro the human periodontal ligament stem cells,in primary culture process by phase contrast microscopy to observe the changes of cell phenotype. Application HE staining and immunohistochemieal study on morphological detection. The cell surfaceantigens such as CD29,CD44,CD105 and CD34, CD45 and HLA-DR were detected by flow eytometry. Induced PDLSCs into osteogenie cells, into fat cells and into neuronal differentiation, and through the specific dyeing analysis, scanning electron micros- copy （sere） to detect the PDLSCs biological activities. Results In vitro successfully isolated and cultured human periodontal ligament stem cells, HE staining showed that cell uniformity; negative expression of keratin and vimentin positive expres- sion, CD44-positive expression. Flow eytometry instrument results showed that PDLSCs surface high expressed CD29 （92.47 ~ ）, CD44（96.42 %）, CD105 （ 96.40 % ）, CD34, CD45 and HLA-DR negative expression. Induced osteogenesis results showed PDLSCs had strong osteogenie activity,alizarin red dyeing results showed that induced group had obvious mineralized nodule formation,ALP staining positive; adipogenic results showed strong adipogenic activity,PDLSCs oil red ＂0＂ fat positive staining;into neurons induced the results showed PDLSCs had strong into neuronal activity,NSE immunohistochemical staining positive expression. Conclusion PDLSCs would have multipotent differentiation potential in vitro.