沉默Versican V0/V1对小鼠牙乳头细胞生物学行为的影响

doi:10.13591/j.cnki.kqyx.2024.05.006

摘要/Abstract

摘要：

目的探究多功能蛋白聚糖(Versican)V0/V1对小鼠牙乳头细胞(mouse dental papilla cells,mDPCs)生物学行为的影响。方法体外培养E16.5d胎鼠mDPCs,使用小干扰RNA(siRNA)沉默mDPCs的Versican V0/V1基因,通过qRT-PCR和免疫荧光染色验证沉默效率;通过EdU实验检测细胞增殖率,划痕实验及Transwell细胞迁移实验检测细胞迁移能力;碱性磷酸酶(ALP)染色和茜素红染色分别评估 mDPCs的矿化情况,并利用 qRT-PCR 检测其成牙和矿化相关基因表达水平。结果 siRNA转染后,与si-NC组相比,si-Versican V0/V1组mDPCs的增殖能力及迁移能力减弱(P<0.01),si-Versican V0/V1组碱性磷酸酶染色更深且矿化结节数量增加,si-Versican V0/V1组成牙和矿化相关的基因表达量增加(P<0.05)。结论沉默Versican V0/V1抑制mDPCs增殖和迁移,但促进mDPCs成牙分化和矿化。

关键词: 多功能蛋白聚糖, 小鼠牙乳头细胞, 细胞增殖, 细胞迁移, 成牙分化

Abstract:

Objective To investigate the effect of Versican V0/V1 on biological behaviors of mouse dental papilla cells (mDPCs). Methods mDPCs were isolated from C57BL/6J mice at embryonic day 16.5 (E16.5). A small interfering RNA (siRNA) constructed specifically for Versican V0/V1 was transfected into mDPCs.The silencing efficiency was verified by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The proliferation rate of mDPCs was determined using EdU assay; the migration ability of mDPCs was evaluated by scratch test and transwell assay. Alkaline phosphatase (ALP) staining and alizarin red staining were used to assess the mineralization capability of mDPCs. The molecules related to odontogenic differentiation and mineralization at mRNA levels were measured by qRT-PCR. Results After siRNA transfection,the mDPCs of si-Versican V0/V1 group showed weaker proliferation and migration abilities compared with si-NC group (P<0.01). An enhanced ALP staining intensity,mineralized nodule formation and up-regulations of the molecules related to odontogenic differentiation and mineralization at mRNA levels (P<0.05) were observed in the mDPCs of si-Versican V0/V1 group. Conclusion Versican V0/V1 silencing inhibits the proliferation and migration of mDPCs,but enhances the abilities of odontogenic differentiation and mineralization.

Key words: Versican, mouse dental papilla cell, cell proliferation, cell migration, odontogenic differentiation

中图分类号:

吴佳艳, 黄海燕, 宋宸宇, 蒋备战. 沉默Versican V0/V1对小鼠牙乳头细胞生物学行为的影响[J]. 口腔医学, 2024, 44(5): 349-355.

WU Jiayan, HUANG Haiyan, SONG Chenyu, JIANG Beizhan. Effects of Versican V0/V1 gene silencing on biological behaviors of mouse dental papilla cells[J]. Stomatology, 2024, 44(5): 349-355.

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基因	引物序列(5'-3')
Versican V0	F:ATGAAGACGGAGAGGAGGACTGTG
Versican V0	R:CACTGTGGTAACGAGCTGCTTCC
Versican V1	F:GCAGATTTGATGCCTACTGC R:ACGGAGTAGTTGTTACATCCG
Alp	F:CACAGACTCCCTGCTTTTGCT R:TGCCTCAGAAGAGCTGAAAACTT
Dspp	F:GTGGGATCATCAGCCAGTCAG R:TGCCTTTGTTGGGACCTTCA
Runx2	F:ATGCATTTAAGATATGGTTGCC R:TGGAGTTGGGAAACACTTGA
Gapdh	F:GACATCAAGAAGGTGGTGAAGC R:GAAGGTGGAAGAGTGGGAGTT