神经生长因子对小鼠颌骨骨髓间充质干细胞增殖、迁移、分化及凋亡的影响

王雅迪; 刘一涵; 张鹤扬; 徐振华; 刘水蓉; 江小霞; 贺慧霞

doi:10.3969/j.issn.2095-5227.2023.05.016

神经生长因子对小鼠颌骨骨髓间充质干细胞增殖、迁移、分化及凋亡的影响

Effects of nerve growth factor on proliferation, migration, differentiation and apoptosis of mouse jaw bone marrow mesenchymal stem cells

摘要

摘要:
背景神经生长因子(nerve growth factor，NGF)是神经营养因子中的一类，能够促进中枢及外周神经元加速生长，修复损伤的神经系统，有研究证实NGF能促进中胚层来源的长骨骨髓间充质干细胞的成骨分化及骨形成，而NGF对外胚层来源的颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells，JBMMSCs)生物学特性的影响尚不明确。
目的探讨NGF对JBMMSCs增殖、迁移、分化及凋亡特性的影响。
方法原代培养小鼠JBMMSCs并鉴定。取第二代JBMMSCs加入含50 ng/mL NGF培养液孵育72h后，通过 CCK-8、qRT-PCR、Western blot及免疫荧光(immunofluorescence，IF)等方法检测细胞增殖及其相关标志物MCM-2、Ki67的表达；qRT-PCR、Western blot检测凋亡相关标志物Bcl-3、Bax的表达；划痕实验检测JBMMSCs迁移能力；碱性磷酸酶(alkaline phosphatase，ALP)染色、qRT-PCR、Western blot及IF检测成骨相关标志物表达。
结果 CCK-8结果显示，50 ng/mL NGF可显著增强JBMMSCs第3天及第5天的OD值，Western blot及IF结果显示，50 ng/mL NGF可显著增强JBMMSCs 中增殖标志物Ki67、MCM-2的蛋白水平；划痕实验结果显示，50 ng/mL NGF可显著提高JBMMSCs的迁移率，有效缩小各个时间点的划痕面积；50 ng/mL NGF可有效增强JBMMSCs的碱性磷酸酶活性，上调ALP、OCN、RUNX2、BMP2的mRNA表达水平及ALP、OCN、BMP2、OPN、RUNX2的蛋白水平；此外，50 ng/mL NGF还可上调Bcl-2的mRNA及蛋白表达水平，下调Bax的mRNA及蛋白表达水平。
结论 50 ng/mL NGF能有效提高JBMMSCs增殖、迁移及成骨分化能力，同时抑制其凋亡。

Abstract:
Background Nerve growth factor (NGF) is one of the neurotrophic factors, which can promote the growth of central and peripheral neurons and repair the damaged nervous system. It has been proved that NGF can promote the osteogenic differentiation and bone formation of mesoderm-derived long bone marrow mesenchymal stem cells. However, the effect of NGF on the biological characteristics of ectoderm-derived jaw bone marrow mesenchymal stem cells (JBMMSCs) remains unclear.
Objective To investigate the effects of NGF on the proliferation, migration, differentiation and apoptosis of JBMMSCs.
Methods Mouse JBMMSCs were cultured and identified. The second generation of JBMMSCs were cultured in medium containing 50 ng/mL of NGF for 72 h. CCK-8, qRT-PCR, Western blot and immunofluorescence (IF) assay and other methods were used to detect the cell proliferation and the expression of related markers MCM-2 and Ki67. qRT-PCR and Western blot were used to detect the expression of apoptosis-related markers Bcl-3 and Bax. The migration ability of JBMMSCs was detected by scratch test. Alkaline phosphatase (ALP) staining, qRT-PCR, Western blot, and IF were used to detect the expression of osteogenic related markers.
Results CCK-8 results showed that 50 ng/mL of NGF could significantly enhance the OD value of JBMMSCs on day 3 and day 5. Western blot and IF results showed that 50 ng/mL of NGF could significantly enhance the protein levels of Ki67 and MCM-2 in JBMMSCs. The results of scratch test showed that 50 ng/mL NGF could significantly increase the migration rate of JBMMSCs and effectively reduce the scratch area at each time point. And it also could effectively enhance the alkaline phosphatase activity of JBMMSCs and up-regulate the mRNA expression levels of ALP, OCN, RUNX2 and BMP2 and the protein levels of ALP, OCN, BMP2, OPN and RUNX2. In addition, 50 ng/mL of NGF could up-regulate the mRNA and protein expression of Bcl-2 and down-regulate the mRNA and protein expression of Bax.
Conclusion It suggests that 50 ng/mL of NGF can effectively promote the proliferation, migration and osteogenic differentiation of JBMMSCs, and inhibit their apoptosis.

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