鹿茸多肽CNT14对口腔黏膜成纤维细胞增殖、迁移的影响

doi:10.19439/j.sjos.2022.06.008

摘要/Abstract

摘要： 目的: 探讨鹿茸多肽CNT14促进口腔黏膜成纤维细胞（human oral mucosa fibroblasth,hOMF）增殖和迁移的作用和相关分子机制。方法: 采用活/死细胞染色评价鹿茸多肽CNT14对hOMF细胞的生物安全性,CCK-8法检测鹿茸多肽CNT14对hOMF细胞增殖的作用,细胞划痕实验检测鹿茸多肽CNT14对hOMF细胞迁移的影响。采用蛋白质免疫印迹检测鹿茸多肽 CNT14刺激后hOMF 细胞内α-SMA、TGF-β1、Smad2和p-Smad2蛋白的情况;Smad2抑制剂对鹿茸多肽CNT14诱导成纤维细胞活化的影响。在新西兰大白兔体内构建角化龈缺损模型,取再生的牙龈组织进行H-E染色;免疫组织化学检测新西兰大白兔再生的牙龈组织中α-SMA、TGF-β1、Smad2和 p-Smad2蛋白的表达量,补充验证鹿茸多肽 CNT14促进口腔牙龈组织再生的能力。采用SPSS 20.0软件包对数据进行统计学分析。结果: 活/死细胞染色实验中,鹿茸多肽CNT14处理hOMF细胞后,细胞存活率在95%以上。鹿茸多肽CNT14刺激hOMF细胞后,与对照组相比,增殖和迁移率显著增加（P<0.05）。鹿茸多肽CNT14刺激hOMF细胞后,细胞内α-SMA、TGF-β1、Smad2和p-Smad2蛋白表达量显著升高（P<0.05）。经Smad2抑制剂阻断鹿茸多肽CNT14诱导成纤维细胞后,细胞内α-SMA表达下降。动物实验中,H-E染色显示,CNT14处理组新西兰大白兔口腔黏膜创口的炎症反应比对照组轻。免疫组织化学染色结果显示,CNT14处理新西兰大白兔牙龈创口后第9天和第11天,再生的牙龈组织中α-SMA、TGF-β1、Smad2和p-Smad2表达量与对照组相比显著升高（P<0.05）。结论: 鹿茸多肽CNT14对hOMF细胞具有良好的生物安全性,能促进口腔黏膜成纤维细胞增殖和迁移,细胞内α-SMA、TGF-β1、Smad2和p-Smad2蛋白表达量升高,有效促进牙龈组织再生。

关键词: 鹿茸多肽, 口腔黏膜成纤维细胞, 角化龈, 再生, 兔

Abstract: PURPOSE: To explore the effect of pilose antler polypeptides CNT14 on proliferation and migration of human oral mucosa fibroblast (hOMF) cells and the related molecular mechanism. METHODS: The biosafety of pilose antler polypeptides CNT14 on hOMF cells was verified by live-dead cell staining kit.CCK-8 assay was used to detect the effect of pilose antler polypeptides CNT14 on hOMF cell proliferation. The effect of pilose antler polypeptides CNT14 on hOMF cell migration was detected by scratch test. Western blot was used to detect the expression of α-SMA, TGF-β1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler polypeptides CNT14. The effect of Smad2 inhibitors on fibroblast activation induced by pilose antler polypeptides CNT14 was evaluated.The model of keratinized gingival defect was established in New Zealand white rabbits, and the regenerated gingival tissue was stained with H-E. The expression levels of α-SMA, TGF-β1, Smad2 and p-Smad2 proteins in the gingival tissues of regenerated New Zealand white rabbits were detected by immunohistochemistry, and the ability of pilose antler polypeptides CNT14 to promote regeneration of oral gingival tissues was verified. Statistical analysis was performed with SPSS 20.0 software package. RESULTS: The survival rate of hOMF cells was above 95% after treated with pilose antler polypeptides CNT14. After stimulation of hOMF cells with pilose antler polypeptides CNT14, the proliferation and migration rates of hOMF cells were increased compared with the control group (P<0.05). The expression of α-SMA, TGF-β1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler peptide CNT14 was increased, and the difference was statistically significant(P<0.05). The expression of α-SMA in fibroblasts induced by Smad2 inhibitor was decreased. In animal experiments, H-E staining showed that the inflammatory response of oral mucosal wounds of New Zealand white rabbits treated with CNT14 was less than that of the control group. Immunohistochemical staining results showed that the expressions of α-SMA, TGF-β1, Smad2 and p-Smad2 in the regenerated gingival tissues of New Zealand white rabbits treated with CNT14 were significantly increased compared with those in the control group on the 9th and 11th days within the gingival wounds(P<0.05). CONCLUSIONS: Pilose antler polypeptides CNT14 has good biosafety and can promote the proliferation and migration of human oral mucosa fibroblast cells, and the expression levels of α-SMA, TGF-β1, Smad2 and p-Smad2 were increased, promoting the regeneration of gingival tissues.

Key words: Pilose antler polypeptides, Human oral mucosa fibroblasts cells, Keratinized gingival, Regeneration, Rabbit

中图分类号:

R781.4

纳飞, 周建忠, 何永文, 谢志刚, 王荃, 李自良. 鹿茸多肽CNT14对口腔黏膜成纤维细胞增殖、迁移的影响[J]. 上海口腔医学, 2022, 31(6): 607-614.

NA Fei, ZHOU Jian-zhong, HE Yong-wen, XIE Zhi-gang, WANG Quan, LI Zi-liang. Pilose antler polypeptide CNT14 promoted proliferation and migration of human oral mucosa fibroblast cells[J]. Shanghai Journal of Stomatology, 2022, 31(6): 607-614.

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