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尼克酰胺对人胚胎干细胞向神经嵴细胞分化的诱导作用
段豪云
李文静
贾艳妮
赵灿
周庆军
李宗义
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20200624-00450
Promoting effect of nicotinamide on generation of neural crest stem cells derived from human embryonic stem cells
Duan Haoyun
Li Wenjing
Jia Yanni
Zhao Can
Zhou Qingjun
Li Zongyi
Authors Info & Affiliations
Duan Haoyun
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
Li Wenjing
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
Jia Yanni
Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250000, China
Zhao Can
Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250000, China
Zhou Qingjun
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
Li Zongyi
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
·
DOI： 10.3760/cma.j.cn115989-20200624-00450
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摘要

目的探讨尼克酰胺(NIC)在人胚胎干细胞(hESCs)诱导分化为神经嵴细胞过程中的作用，为hESCs进一步分化为角膜内皮细胞提供实验基础。

方法取培养5~7 d的hESCs细胞系H1进行诱导，根据诱导培养基中干预组分的不同分为未添加NIC组(仅使用诱导培养基)、NIC添加组(含10 mmol/L NIC的诱导培养基)、NIC+白藜芦醇(Res)组(含10 mmol/L NIC、10 μmol/L Res的诱导培养基)和Sirtinol组(含10 μmol/L Sirtinol的诱导培养基)，其中Res和Sirtinol分别为SIRT1活性激动剂和抑制剂，各组连续诱导7 d。采用实时荧光定量PCR法检测诱导1、3、5、7 d hESCs及神经嵴细胞标志物mRNA相对表达量；诱导后7 d，采用免疫荧光染色法测定hESCs来源神经嵴细胞相关标志蛋白的表达，采用流式细胞术分析hESCs来源神经嵴细胞表面标志物神经生长因子受体(P75)和人自然杀伤因子1(HNK-1)阳性细胞比率，以评价NIC诱导效率及调控SIRT1对HNK-1表达的影响。

结果与未添加NIC组相比，NIC添加组处理5 d后全能性基因八聚体结合转录因子4(OCT4)和同源域蛋白(NANOG) mRNA相对表达量显著降低，神经嵴细胞标志物P75、HNK-1、SRY相关HMG盒9(SOX9)和SOX10 mRNA相对表达量显著升高，差异均有统计学意义(均 P<0.05)。免疫荧光染色结果显示，未添加NIC组神经嵴细胞标志物P75表达较弱，HNK-1仅有零星表达，转录因子激活蛋白2β(AP-2β)及成对样同源域转录因子2(PITX2)均未见阳性着色；NIC添加组细胞中P75、HNK-1、AP-2β及PITX2均呈广泛的强阳性表达。NIC添加组P75 +HNK-1 +和P75 +细胞比率明显高于未添加NIC组，差异均有统计学意义( t＝8.481， P＝0.001； t＝2.987， P＝0.041)。未添加NIC组、NIC添加组、NIC+Res组和Sirtinol组HNK-1 +细胞比率分别为(34.267±12.522)%、(89.633±1.358)%、(64.667±6.429)%和(86.300±3.460)%，总体比较差异有统计学意义( F＝36.799， P<0.001)，其中，NIC+Res组HNK-1 +细胞比率均明显低于NIC添加组和Sirtinol组，差异均有统计学意义(均 P<0.05)。

结论NIC可通过抑制SIRT1的活性促进HNK-1表达，提高hESCs来源神经嵴细胞的分化效率，为神经嵴细胞相关疾病的治疗，如角膜内皮移植提供新的种子细胞来源。

神经嵴;尼克酰胺;诱导分化;人胚胎干细胞
ABSTRACT

ObjectiveTo investigate the role of nicotinamide (NIC) in the differentiation of neural crest cells from human embryonic stem cells (hESCs), and lay the foundation for the induction of hESC-derived corneal endothelial cells.

MethodshESCs line H1 cultured for 5-7 days was used for induction.According to the different components of the neural crest induction medium, cells were assigned into different groups for 7-days induction, including group treated without NIC cultured in induction medium only, group treated with NIC cultured in induction medium containing 10 mmol/L NIC, NIC+ resveratrol (Res) group cultured in induction medium containing 10 mmol/L NIC and 10 μmol/L Res and Sirtinol group cultured in induction medium containing 10 μmol/L Sirtinol.Res and Sirtinol were used as SIRT1 activity agonist and inhibitor, respectively.The relative mRNA expression levels of hESCs and neural crest cell markers were detected by real-time fluorescence quantitative PCR at 1, 3, 5 and 7 days during the induction.The expression of neural crest cells markers after 7 days of induction was assayed by immunofluorescence staining.The induction efficiency of NIC and the effect of SIRT1 regulation on human natural killer 1 (HNK-1) positive cells expression were evaluated through flow cytometry analysis of percentages of nerve growth factor receptor (P75) and HNK-1 + cells.

ResultsCompared with the group treated without NIC, the mRNA expressions of totipotent genes octamer transcription factor 4 (OCT4) and homeodomain proteins (NANOG) were significantly decreased, and the mRNA expression levels of neural crest cell markers P75, HNK-1, SRY-related HMG box (SOX) 9 and SOX10 were significantly increased in the group treated with NIC after 5 days of induction (all at P<0.05). In the group treated without NIC, P75 was weakly expressed, and HNK-1 was sporadically expressed, and transcription factor AP-2β (AP-2β) and paired-like homeodomain transcription factor 2 (PITX2) were not detected.In the group treated with NIC, P75, HNK-1, AP-2β and PITX2 were strongly expressed.The proportion of P75 + HNK-1 + cells and P75 + cells were both significantly higher in the group treated with NIC than without NIC ( t=8.481, P=0.001; t=2.987, P=0.041). The percentage of HNK-1 + cells in groups treated without and with NIC, NIC+ Res group and Sirtinol group were (34.267±12.522)%, (89.633±1.358)%, (64.667±6.429)% and (86.300±3.460)%, respectively, with a statistically significant overall difference ( F=36.799, P<0.001). The proportion of HNK-1 + cells in NIC+ Res group was significantly lower than that in the groups treated with NIC and Sirtinol (all at P<0.05).

ConclusionsNIC promotes the differentiation of hESCs-derived neural crest cells by inhibiting the activity of SIRT1 to enhance the expression of HNK-1.NIC treatment may provide a new strategy for source of seed cells in the treatment of neural crest cell-related diseases, such as corneal endothelial transplantation.

Neural crest;Nicotinamide;Cell differentiation;Human embryonic stem cells
Li Zongyi, Email: mocdef.3ab61911iygnozil
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引用本文

段豪云,李文静,贾艳妮,等. 尼克酰胺对人胚胎干细胞向神经嵴细胞分化的诱导作用[J]. 中华实验眼科杂志,2022,40(12)：1141-1148.

DOI:10.3760/cma.j.cn115989-20200624-00450

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角膜移植是当前临床治疗角膜内皮病变和内皮失代偿的首选方式，然而角膜供体的严重匮乏制约了其临床应用。2018年，研究者利用培养的原代人角膜内皮细胞对大疱性角膜病变患者进行注射移植获得成功，为角膜内皮功能失代偿的治疗提供了新途径 [ 1 ]。然而，该方法仍不能摆脱对供体材料的需求 [ 2 ]。因此，寻找其他的细胞来源仍是目前的研究热点之一。在脊椎动物眼胚胎发育过程中，角膜内皮的形成源于颅面部神经嵴细胞的迁移和分化 [ 3 , 4 ]。人胚胎干细胞(human embryonic stem cells，hESCs)因具有无限的自我更新和多向分化能力成为良好的种子细胞来源 [ 5 , 6 ]。将hESCs诱导分化为神经嵴细胞，再进一步诱导分化为角膜内皮细胞是获得足量内皮细胞的有效方法。采用饲养层细胞共培养或条件培养基等方式诱导hESCs分化模拟了体内发育过程 [ 7 , 8 ]，然而诱导体系成分的不确定极易导致细胞异质性的产生，且诱导时间长、神经嵴细胞诱导效率低 [ 9 ]，影响了目的细胞的获得。随着神经嵴分化过程中信号通路的研究，对BMP/SMAD、WNT、转化生长因子β(transforming growth factor β，TGF-β)等信号通路进行调控，在无饲养层条件下利用小分子化合物，如糖原合成酶激酶3、TGF-β抑制剂能够促使hESCs由神经外胚层向神经嵴细胞分化 [ 10 , 11 , 12 ]。此外，联合生长因子及多种信号通路抑制剂的"鸡尾酒"体系也被用于神经嵴细胞的诱导 [ 13 , 14 ]。但上述方法仍存在诱导时间较长、诱导体系复杂、可能伴随其他神经外胚层细胞混杂、诱导试剂费用昂贵等问题 [ 15 , 16 ]。进一步简化诱导体系，经济、高效获得较高纯度的hESCs来源神经嵴细胞将极大促进角膜内皮再生领域的发展。尼克酰胺(nicotinamide，NIC)可用于干细胞重编程，促进并维持干细胞向视网膜色素上皮、神经、胰腺等不同谱系分化，是维持干细胞多能性、调节诱导分化的重要因子 [ 6 , 17 , 18 , 19 , 20 ]。本研究拟探讨NIC在hESCs诱导分化为神经嵴细胞过程中的作用，以期为hESCs进一步分化为角膜内皮细胞提供实验基础。
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A
李宗义，Email： mocdef.3ab61911iygnozil
B

段豪云：参与设计实验、实施研究、采集/分析数据、起草及修改文章；李文静、贾艳妮、赵灿：实施研究、采集/分析数据；周庆军：参与选题的确定；李宗义：参与实验设计的把控、文章审阅及定稿

C
感谢陆军军医大学第一附属医院眼科阴正勤教授提供H1细胞系
D
所有作者均声明不存在利益冲突
E
国家自然科学基金项目 (81700811、81900834)
山东省自然科学基金项目 (ZR2018LH008)
山东第一医科大学学术提升计划项目 (2019RC008)
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