Effect of cannabinoid type I receptors on neuronal differentiation of human apical papilla stem cells

doi:10.12307/2026.509

Abstract

Abstract: BACKGROUND: Previous studies have demonstrated that the cannabinoid type I receptor can enhance the proliferation and neural differentiation of neural stem cells and mesenchymal stem cells. Moreover, cannabinoid type I also governs the proliferation and mineralization capacity of human apical papilla stem cells. However, there are relatively few investigations concerning the impact of cannabinoid type I overexpression on the neural differentiation of human apical papilla stem cells.
OBJECTIVE: To investigate the effect of cannabinoid type I on neural differentiation of human apical papilla stem cells in vitro.
METHODS: Healthy third molars with immature root tips that need to be removed for orthodontic treatment were collected, and human apical papilla stem cells were isolated and cultured by tissue block method combined with enzyme digestion method. Cannabinoid type I gene was introduced into human apical papilla stem cells by lentivirus-mediated transfection technique. A blank control group, a negative control group, and cannabinoid type I overexpression group were set up. The transfection effect of overexpression of cannabinoid type I lentivirus on human apical papilla stem cells was verified by Western Blot. The control group, negative control group, cannabinoid type I overexpression group and cannabinoid type I overexpression + AM251 (cannabinoid type I receptor antagonist) group were set up. Cell proliferation was detected by CCK-8 assay at 1, 5, and 10 days after neural induction. On day 10 of neural induction, the expression levels of TH, NeuroD-1, and NCAM1 genes were detected by qRT-PCR, and the protein expression levels of Nestin and TUBB3 were detected by immunofluorescence.
RESULTS AND CONCLUSION: (1) Compared with the blank control group and the negative control group, the expression of cannabinoid receptor I protein in the cannabinoid receptor I overexpression group was significantly increased, and the difference was significant (P < 0.05). (2) Compared with the blank control group and the negative control group, the proliferation ability of human apical papilla stem cells in the cannabinoid type I overexpression group was the strongest at 5 and 10 days after neural induction (P < 0.05). (3) Compared with the blank control group and the negative control group, the mRNA expression of NeuroD-1, NCAM1, and TH in the stem cells of the human apical papilla in the cannabinoid type I overexpression group was significantly increased, and the fluorescence intensity of Nestin and TUBB3 was significantly enhanced (P < 0.05). (4) Compared with the cannabinoid type I overexpression group, the proliferation ability, mRNA expression level of NeuroD-1, NCAM1, and TH, as well as the fluorescence intensity of Nestin and TUBB3, were significantly decreased in the cannabinoid type I overexpression + AM251 group (P < 0.05). These findings conclude that overexpression of cannabinoid type I promoted the proliferation and neural differentiation of human apical dentin papilla stem cells.

Key words: ">human apical dental papilla stem cell, dental papilla, cannabinoid type I receptor, neural differentiation, proliferation, lentivirus, AM251, nerve injury

CLC Number:

Liu Ziwei, Nijati·Tursun, Yin Rui, Li Shuhui, Zhou Jing. Effect of cannabinoid type I receptors on neuronal differentiation of human apical papilla stem cells[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(1): 93-100.

Figures/Tables 3

2.1 人根尖牙乳头干细胞形态组织取材自新鲜拔除的牙根尚未发育完全的恒牙根尖部，酶消化法联合组织块法培养原代根尖牙乳头干细胞，培养3 d后发现培养瓶底部有少量不规则多角形细胞爬出(图1A)，培养5 d后可见细胞数量增加，中央有卵圆形核，胞质向外伸出长短不同的突起(图1B)，培养7 d后细胞呈现均一的长梭形(图1C)，培养10 d后大量细胞聚集，相互紧密连接(图1D)。细胞生长速度快，融合度在70%-80%时，胰酶消化传代，扩大培养。 2.2 人根尖牙乳头干细胞流式鉴定结果对人根尖牙乳头干细胞进行流式细胞术分析，结果显示，间充质干细胞标志物CD24、CD146和STRO-1表达率分别为93.5%，44.29%和35.79%，而造血干细胞标志物CD45表达率为0.00%，见图2。 2.3 慢病毒最佳感染条件荧光显微镜下观察荧光强度，与其他两组相比，感染72 h后，HitransG A感染增强液、感染复数=10条件下，人根尖牙乳头干细胞感染效率达80%以上，荧光亮度表达最强，且细胞生长状况良好，见图3。因此，选取该条件进行后续慢病毒感染实验。 2.4 Western blot检测人根尖牙乳头干细胞内CB1的表达与阴性对照组及空白对照组相比，过表达CB1组CB1蛋白表达量明显升高，差异有显著性意义(P < 0.05)。空白对照组与阴性对照组的CB1蛋白表达量无显著差异(P > 0.05)，见图4。 2.5 细胞增殖检测结果 CCK-8检测结果显示：人根尖牙乳头干细胞成神经诱导第1天时，各组间吸光度值无明显差异。人根尖牙乳头干细胞成神经诱导第5，10天时，与空白对照组及阴性对照组相比，过表达CB1组人根尖牙乳头干细胞的增殖能力显著升高(P < 0.05)；与过表达CB1组相比，过表达CB1+AM251组人根尖牙乳头干细胞的增殖能力显著下降(P < 0.05)，见图5，提示CB1基因过表达促进人根尖牙乳头干细胞的增殖，AM251的加入抑制了人根尖牙乳头干细胞的增殖。 2.6 各组人根尖牙乳头干细胞形态神经分化诱导1 d时可见少数具有细长形突起的星状细胞，细胞分布较为分散，各组间未见显著差异。神经分化诱导5 d时，与阴性对照组及空白对照组相比，过表达CB1组的细胞数量明显增多，细胞突起长度增长，分支数量增多，突起之间形成网络状连接；神经分化诱导10 d时，细胞增殖速度有所下降，细胞形态由梭形转变为圆形，形成了具有多极突起的典型神经细胞形态，表明CB1基因可促进人根尖牙乳头干细胞神经突起的生长。神经分化诱导第5，10天时，与过表达CB1相比，过表达CB1+AM251组细胞数量减少，胞质向外伸出两三个长短不同的突起，细胞间连接较为松散，神经细胞样形态不明显，提示AM251抑制人根尖牙乳头干细胞向神经细胞分化，见图6。"

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