含毛喉素的条件培养液诱导脂肪来源干细胞神经分化的体外实验研究

doi:10.12439/kqhm.1005-4979.2024.03.002

摘要/Abstract

摘要：

目的: 利用神经诱导液体外诱导大鼠脂肪来源干细胞（adipose-derived stem cells，ASCs）神经分化，探索对ASCs体外成神经分化的最佳毛喉素浓度，并研究该浓度的培养液对ASCs体外成神经分化的作用。方法: 酶消化法获得原代ASCs，通过流式细胞术和细胞染色鉴定ASCs的表型和多向分化能力。体外用含不同浓度毛喉素的神经诱导条件培养液进行神经诱导分化，采用免疫荧光染色技术筛选最佳浓度。采用含最佳浓度毛喉素的神经诱导条件培养液诱导ASCs，镜下观察诱导前后细胞的形态学特征，采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR)和免疫荧光检测诱导前后细胞中神经分化标志物的mRNA和蛋白表达变化情况。结果: 神经诱导条件培养液中毛喉素的最佳浓度为10 μmol/L，含该浓度毛喉素的神经诱导条件培养液诱导的细胞，能够迅速诱导ASCs向成神经方向分化。诱导后，细胞形态类似神经细胞，神经细胞标志物抗β-Ⅲ微管蛋白（class Ⅲ β-tubulin，Tuj1）、神经丝蛋白（neurofilament-M，NF-M）、巢蛋白（nestin）抗体和S100β的mRNA水平和蛋白水平均表达增加（P<0.05）。结论: 含毛喉素浓度为10 μmol/L的诱导培养液能诱导ASCs神经分化，诱导细胞有望成为周围神经种子细胞。

关键词: 脂肪来源干细胞, 细胞分化, 神经再生, 毛喉素

Abstract:

Objective: To explore the neural differentiation of adipose-derived stem cells (ASCs) induced by neural-induced conditional medium in vitro, and to study the optimal concentration of forskolin and the effect of optimal concentration on neurogenic differentiation of ASCs in vitro. Methods: ASCs were isolated by enzyme digestion method. The phenotype and differentiation ability of stem cells are identified by flow cytometry and cell staining. Nerve induction differentiation was performed in vitro by neural-induced conditional medium containing different concentrations of forskolin, and the optimal concentration was screened by immunofluorescence staining. ASCs were induced by neural-induced conditional medium containing the optimal concentration of forskolin and the morphological changes of cells were observed by microscope before induction, 6 h after induction and 24 h after induction. Effects of induction on rat ASCs were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence. Results: The optimal concentration of forskolin in neural-induced conditional medium was 10 μmol/L, and the cells induced by neural-induced conditional medium containing 10 μmol/L forskolin could rapidly induce ASCs to differentiate in the direction of nerve formation. After induction, the cell morphology was similar to nerve cells. Results of RT-qPCR and immunofluorescence indicated that the expressions of anti-class Ⅲ β-tubulin (Tuj1), neurofilament-M (NF-M), nestin and S100 β at the mRNA level and protein level were increased after induction (P<0.05). Conclusion: Nerve differentiation of ASCs can be induced by conditional medium containing 10 μmol/L forskolin, and the induced cells are expected to become seed cells of facial nerve conduits.

Key words: adipose-derived stem cells, cell differentiation, nerve regeneration, forskolin

中图分类号:

R782

刘旭凌, 胡颖涵, 孙家悦, 朱泽宇, 陆家瑜. 含毛喉素的条件培养液诱导脂肪来源干细胞神经分化的体外实验研究[J]. 《口腔颌面外科杂志》, 2024, 34(3): 170-179.

LIU Xuling, HU Yinghan, SUN Jiayue, ZHU Zeyu, LU Jiayu. Forskolin-containing conditioned medium induces neural differentiation of adipose-derived stem cells: An in vitro experimental study[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(3): 170-179.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I3/170

图/表 9

表1 基因引物序列

Table 1 Sequences of primers

基因	引物序列(5′-3′)	引物序列(3′-5′)
NF-M	GTGGTTCAAATGCCGCTACG	GCTGGATGGTGTCCTGGTAG
Tuj1	CAACTATGTGGGGGACTCGG	TGGCTCTGGGCACATACTTG
S100 β	AGAGGGTGACAAGCACAAGC	TTGTCCACCACTTCCTGCTC
nestin	AGCACTCCCATCCCACCTAT	GGGTTGTGGCTAAGGAGGTC
GAPDH	GCCAGTAGACTCCACGACA	GCAAGTTCAACGGACAAG

图1 大鼠ASCs的形态学观察和鉴定（×40） A. ASCs培养12 h的贴壁细胞；B. ASCs培养5~7 d的贴壁细胞；C. P3 ASCs能够阳性表达CD29；D. P3 ASCs能够阳性表达CD90；E. P3 ASCs能够阴性表达CD11b；F. P3 ASCs能够阴性表达CD45。

Figure 1 Morphological observation and identification of rat ASCs（×40）

图2 大鼠ASCs多向分化能力检测（×100） A. P3 ASCs油红O染色观察成脂分化效果；B. P3 ASCs茜素红染色观察成骨分化效果；C. P3 ASCs番红O-固绿染色观察成软骨分化效果。

Figure 2 The multi-lineage differentiation capacity of rat ASCs（×100）

图3 MTT法检测第3代ASCs的生长曲线

Figure 3 Growth curves of P3 ASCs detected by MTT assay

图4 免疫荧光染色鉴定不同浓度毛喉素的诱导效果（×200） A.不含毛喉素的神经诱导条件培养液诱导ASCs神经元向分化的效果；B—E.毛喉素浓度分别为5、10、15、20 μmol/L的神经诱导条件培养液诱导ASCs神经元向分化的效果。

Figure 4 Immunofluorescence detection of cells induced by different concentrations of forskolin（×200）

图5 诱导细胞的形态学观察（×100） A. P3 ASCs贴壁细胞；B.神经诱导6 h后的细胞；C.神经诱导24 h后的细胞。

Figure 5 Morphological observation of induced cells (×100)

图6 免疫荧光染色鉴定诱导前后的细胞（×200） A. P3 ASCs的nestin、S100 β和Tuj1荧光染色效果；B.神经诱导6 h后细胞nestin、S100 β和Tuj1的荧光染色效果；C.神经诱导24 h后细胞nestin、S100 β和Tuj1的荧光染色效果。

Figure 6 Immunofluorescence detection of ASCs and induced cells (×200)

图7 Tuj1阳性的神经元的量化分析 A.从左至右依次为神经诱导6 h的细胞Tuj1荧光染色图（×200，白色虚线框为随机选择的视野）、形态观察及量化分析；B.从左至右依次为神经诱导24 h的细胞Tuj1荧光染色图（×200，白色虚线框为随机选择的视野）、形态观察及量化分析；C.诱导前后Tuj1染色阳性的神经元分支的数量；D.神经突总长度；E.诱导不同时间的Tuj1染色阳性神经元的分支分布。***表示P<0.001，与对照组（ASCs）比较。

Figure 7 Quantification analysis of Tuj1-positive neurons

图8 RT-qPCR检测诱导前后大鼠ASCs中Tuj1、nestin、S100 β和NF-M的mRNA表达 A. Tuj1的mRNA表达；B. nestin的mRNA表达；C. S100 β的mRNA表达；D. NF-M的mRNA表达。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 8 The mRNA expression of Tuj1、Nestin、S100 β and NF-M in ASCs and induced cells using RT-qPCR

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