摘要目的 探讨人乳牙牙髓干细胞(stem cells from the pulp of human exfoliated deciduous teeth,SHED)和恒牙牙髓干细胞(dental pulp stem cells,DPSC)的一般生物学特性及与唾液腺分泌相关分子的表达差异,为使用SHED或DPSC治疗唾液腺分泌功能低下提供依据.方法 分别培养SHED和DPSC至第4、7代,使用组织块法原代培养人下颌下腺上皮和间质细胞,使用相差显微镜观察细胞形态;使用流式细胞术检测干细胞表面标志物;分别使用细胞计数试剂盒(cell counting kit-8,CCK-8)、长时间动态活细胞成像及数据分析系统IncuCyte ZOOM检测细胞增殖;使用实时荧光定量PCR检测与分泌相关的分子表达.结果 第4和7代SHED及DPSC均呈长梭形贴壁生长.4组细胞均阳性表达间充质干细胞表面标志物CD29、CD44、CD73和CD90,阴性表达上皮干细胞标志物CD49f和CD117.4组细胞增殖无差异.与第4代SHED相比,第7代SHED中与水分泌相关的毒蕈碱型乙酰胆碱受体1(muscarinic acetylcholine receptor 1,MR1)和MR3、水通道蛋白5(aquaporin 5,AQP5),与蛋白质分泌相关的β1-肾上腺素受体(β1-adrenoceptor,β1-AR)及分泌蛋白α-淀粉酶和黏蛋白5B的表达差异均无统计学意义(P>0.05),β2-AR的表达显著降低(P<0.05).与第4代DPSC相比,第7代DPSC中MR3、β2-AR和α-淀粉酶表达差异无统计学意义(P>0.05),而MR1、AQP5、β1-AR和黏蛋白5B表达降低(P<0.05).与1例同年龄段人的下颌下腺组织及其原代上皮细胞相比,第4和7代SHED中各分子表达均低.与同年龄段人的下颌下腺原代上皮细胞相比,第4代DPSC中AQP5表达显著降低(P<0.05),其他分子表达差异无统计学意义(P>0.05);第7代DPSC中AQP5、β1-AR、α-淀粉酶和黏蛋白5B表达显著降低(P<0.05),其他分子表达差异无统计学意义(P>0.05).第4和7代DPSC中各分子的表达均显著低于人下颌下腺组织中的表达(P<0.01).结论 与DPSC相比,SHED生长稳定,与唾液腺水和蛋白质分泌相关的分子表达较低;SHED可成为唾液腺组织工程及治疗唾液腺分泌功能低下的种子细胞,且第4至7代SHED均可用于实验研究.

abstractsObjective To compare the general biological characteristics and the expressions of proteins involved in secretion in stem cells from the pulp of human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC). Methods SHED and DPSC were cultured and collected at passage 4 (P4)and P7. The submandibular gland epithelial and interstitial cells were cultured with tissue culture method. The cell morphology was observed using a phase contrast microscope. Flow cytometry was used to detect stem cell surface markers. Cell counting kit-8 (CCK-8) and IncuCyte ZOOM were used to evaluate cell proliferation. Quantitative real-time PCR (qPCR) was performed to examine the mRNA expressions of proteins involved in fluid and protein secretion. Results P4 and P7 SHED and DPSC were spindle-shaped. There was no difference in cell morphology among the 4 group cells. P4 and P7 SHED and DPSC expressed CD29, CD44, CD73, and CD90, the mesenchymal stem cell markers, while, CD49f and CD117, the epithelium markers were undetected. There was no difference in cell proliferation among the 4 group cells. Compared with P4 SHED, the expressions of muscarinic cholinergic receptor 1 (MR1), MR3, aquaporin 5 (AQP5),β1-adrenoceptor (β1-AR),α-amylase, and mucin 5B in SHED were not different, whileβ2-AR expression was decreased (P<0.05). Compared with P4 DPSC, the expressions of MR3,β2-AR, andα-amylase in P7 DPSC were not different, while, the expressions of MR1, AQP5,β1-AR, and mucin 5B were decreased (P<0.05). Compared with primary cultured submandibular gland epithelial cells and gland tissues from a child, the expressions of proteins involved in secretion were all decreased. Compared with submandibular epithelial cells from adults, the expression of AQP5 in P4 DPSC was decreased (P<0.05), while other proteins were not different. The expressions of AQP5,β1-AR,α-amylase and mucin 5B in P7 DPSC were increased (P<0.05), while other proteins were not different. In P4 and P7 DPSC, all the protein expression levels were decreased, compared with those in submandibular gland tissues (P<0.01). Conclusions Compared with DPSC, SHED have stable growth and the expressions of protein involved fluid and protein secretion are low. Based on its extensive sources and easy separation, SHED can be used as the ideal seed cell for salivary gland tissue engineering and the treatment of salivary gland hypofunction, and the P4 to P7 SHED can be used for experimental study.