摘要
目的:探究内源性透明质酸(hyaluronan,HA)对小鼠牙乳头细胞(mouse dental papilla cells,mDPCs)增殖的影响.方法:对出生后2.5 d的小鼠腹腔注射5-乙炔基-2'脱氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,EdU)标记快速增殖细胞;体外分离培养mDPCs,使用不同浓度透明质酸酶(hyaluronidase,HAase)降解HA,细胞计数试剂盒(cell counting kit-8,CCK-8)法及EdU法检测细胞增殖;使用流式细胞术检测细胞周期分布和凋亡;转染siHyal2抑制HA降解,EdU法检测细胞增殖.结果:HA主要表达在小鼠切牙根尖慢增殖间充质细胞周围,而在转运扩增细胞(transit-amplifying cells,TACs)表达下降;CCK-8结果显示,相比对照组,800 µg/mL HAase明显促进细胞增殖(P＜0.0001);EdU检测及流式细胞术结果显示,HAase处理48 h后,细胞DNA合成增加,S期细胞比率明显增加(P＜0.01),并抑制早期细胞凋亡(P＜0.05);与之相反,当HA积聚在mDPCs细胞周,细胞增殖受到抑制(P＜0.01).结论:内源性HA与mDPCs增殖密切相关,HA分解可促进细胞增殖;而局部HA的积聚则抑制细胞增殖.
Abstract
Objective:To explore the effect of endogenous hyaluronan(HA)remodeling on the proliferation of mouse dental papilla cells(mDPCs).Methods:Fast-proliferating cells were labeled with EdU by intraperitoneal in-jection into mice at postnatal day 2.5.mDPCs were isolated,and then treated with different concentrations of hyal-uronidase(HAase).The cell proliferation was detected by CCK-8 assay and EdU assay.Flow cytometry was used to detect cell cycle distribution and apoptosis.The transfection of siHyal2 caused HA accumulation and the cell proliferation was detected by EdU assay.Results:HA expression was mainly observed in the slow-proliferating mesenchymal cells in mouse incisors,while the expression of HA was decreased in transit-amplifying cells(TACs).The CCK-8 results indicated that 800 μg/mL HAase significantly promoted cell proliferation compared with the con-trol group(P＜0.0001).After 48 h of HAase treatment,the EdU assay and flow cytometry results showed an in-crease in cellular DNA synthesis and a significant increase in the ratio of S-phase cells(P＜0.01).Additionally,early cell apoptosis was inhibited(P＜0.05).In contrast,the accumulation of HA around mDPCs was found to sig-nificantly inhibit cell proliferation(P＜0.01).Conclusion:The proliferation of mDPCs is closely related to endoge-nous HA.The degradation of HA promotes cell proliferation,which is inhibited by local HA accumulation.
基金项目
国家自然科学基金(8230035085)
上海市卫生健康委员会科研项目(202140357)
上海申康医院发展中心新兴前沿技术联合攻关项目(SHDC12023115)
NETL
NSTL
万方数据