不同细胞标记物对大鼠牙髓干细胞体外标记的生物学影响  

Effects of different cell markers on biological characters rat dental pulp stem cells in vitro

作　　者：谢娜 姬小婷 李子夏 唐成芳 陆磊 王丹杨 XIE Na;JI Xiaoting;LI Zixia;TANG Chengfang;LU Lei;WANG Danyang(Department of Oral Medicine,School of Stomatology,Xi’an Medical University,Xi’an 710021,China;Department of Stomatology,Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine;Department of Prosthodontics,School of Stomatology,Xi’an Medical University)

机构地区：[1]西安医学院口腔医学院口腔内科学教研室,西安710021 [2]陕西中医药大学附属医院口腔科 [3]西安医学院口腔医学院口腔修复学教研室

出　　处：《山西医科大学学报》2021年第6期735-740,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金青年科学基金项目(81701014);陕西省自然科学基础研究计划面上项目(2017JM8038);西安医学院校级配套基金项目(2018PT29)。

摘　　要：目的通过体外实验,对比氯甲基苯甲酰氨(CM-DiI)和4′,6-二脒基-2-苯基吲哚(DAPI)这两种不同细胞标记物对大鼠牙髓干细胞生物学性能的影响,为需要进行细胞标记的实验研究提供理论参考依据。方法采用酶解组织块法培养大鼠牙髓细胞并对其进行纯化,通过免疫组化鉴定细胞来源,并进行细胞体外多向诱导分化能力实验。利用5μg/ml浓度的CM-DiI和DAPI对第3代大鼠牙髓干细胞进行体外标记,通过比较细胞增殖情况以及细胞乳酸脱氢酶释放率,评价标记后细胞生物学状态。结果利用酶解组织块法可获得克隆集落状生长的大鼠牙髓细胞。免疫组化法确定细胞为间充质来源,且具有向脂肪细胞、成骨细胞以及成牙本质细胞分化的潜能。CM-DiI标记后细胞膜及核膜均呈现出红色荧光,DAPI则细胞核蓝染。与未进行细胞标记比较,CM-DiI和DAPI标记后细胞形态、上清液乳酸脱氢酶含量及CCK-8值均无明显变化,两组差异无统计学意义。结论CM-DiI和DAPI操作简便、染色速度快,都可用于标记大鼠牙髓干细胞。相较DAPI而言,CM-DiI对大鼠牙髓干细胞的细胞毒性更小。Objective To compare the effects of CM-DiI and DAPI on the biological properties of rat dental pulp stem cells in vitro for providing a theoretical basis for the experimental study of cell labeling.Methods Enzyme tissue block method was used to culture dental pulp stem cells from Sprague-Dawley rats,and the biological characteristics of cells were identified.The third generation rat dental pulp stem cells were labeled with 5μg/ml CM-DiI and DAPI in vitro.The release of lactic dehydrogenase(LDH)and cell proliferation were used to evaluate the biological properties of cells in vitro.Results The rat dental pulp cells were harvested by enzyme tissue block method.Immunohistochemistry staining confirmed that the cells were derived from mesenchymal sources,and had the potential to differentiate into adipocytes,osteoblasts and odontoblasts.After CM-DiI labeling,the cell membrane and nuclear membrane showed red fluorescence,while DAPI results showed the cell nucleus was blue staining.Compared with that without cell labeling,there were no significant changes in cell morphology,supernatant lactate dehydrogenase content and CCK-8 values after CM-DiI and DAPI labeling.Conclusion CM-DiI and DAPI could be used to label rat dental pulp stem cells.Compared with DAPI,CM-DiI has less cytotoxicity to rat dental pulp stem cells.

关 键 词：牙髓干细胞 细胞标记 CM-DiI DAPI 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]