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Article(id=1320442409910129339, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442360509616480, articleNumber=null, orderNo=null, doi=10.16016/j.2097-0927.202601032, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1768060800000, receivedDateStr=2026-01-11, revisedDate=1774454400000, revisedDateStr=2026-03-26, acceptedDate=null, acceptedDateStr=null, onlineDate=1792747603155, onlineDateStr=2026-10-23, pubDate=1778774400000, pubDateStr=2026-05-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792747603155, onlineIssueDateStr=2026-10-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792747603155, creator=13701087609, updateTime=1792747603155, updator=13701087609, issue=Issue{id=1320442360509616480, tenantId=1146029695717560320, journalId=1319373162502258839, year='2026', volume='48', issue='9', pageStart='1129', pageEnd='1288', issueExtLink='null', onlineDate='null', pubDate='1778774400000', pubDateStr='2026-05-15', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1792747591378, creator='13701087609', updateTime=1792749835358, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1320451772548760069, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442360509616480, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1320451772548760070, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442360509616480, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1181, endPage=1193, ext={EN=ArticleExt(id=1320442410132427452, articleId=1320442409910129339, tenantId=1146029695717560320, journalId=1319373162502258839, language=EN, title=Spheroid culture induces a quiescent state in nucleus pulposus stem cells by inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy, columnId=1320434418116837551, journalTitle=Journal of Army Medical University, columnName=Basic Medicine, runingTitle=null, highlight=null, articleAbstract=
Objective

Intervertebral disc degeneration (IVDD) is a primary cause of low back pain, and stemness exhaustion of nucleus pulposus stem cells (NPSCs) represents a key underlying mechanism. Thus, maintaining the quiescent state of NPSCs is of great significance in delaying disc degeneration. This study aimed to investigate the inductive effect of in vitro three‑dimensional (3D) spheroid culture on the quiescent state of NPSCs and its regulatory mechanism, thereby providing experimental evidence for targeted intervention in IVDD.

Methods

Intervertebral disc tissues were collected from juvenile and adult patients undergoing lumbar surgery in our department between December 2025 and January 2026. Tissue sections were prepared, and morphological differences were observed through HE staining. NPSCs were isolated from 24 male SD rats (8 weeks old, weighing 350±50 g). Stem cell immunophenotypes were identified by flow cytometry, and the stemness of NPSCs was verified by trilineage differentiation assay. NPSCs cultured under routine 2D conditions served as the monolayer culture (ML) group, while NPSCs cultured using the 3D spheroid culture system were established as the spheroid culture (SP) group. Cell morphology was observed under a microscope, cell cycle distribution was analyzed by flow cytometry, and the quiescent state was identified by EdU staining. Bulk transcriptome sequencing and bioinformatic analysis were performed on NPSCs from the ML and SP groups. The expression of autophagy‑related molecules and key molecules in the PI3K‑Akt‑mTOR signaling pathway, as well as the ultrastructure of autolysosomes, were detected by RT‑qPCR, Western blotting and transmission electron microscopy (TEM), respectively.

Results

Clinical histological observation showed that nucleus pulposus cells in juvenile patients were scattered in distribution, whereas those in adult patients exhibited clustered distribution, and the proportion of clustered nucleus pulposus cells was significantly higher in adult patients than in juvenile patients(P<0.01). The successfully isolated rat NPSCs highly expressed mesenchymal stem cell‑positive markers CD90 (99.4%), CD44 (99.8%) and CD73 (94.2%), and lowly expressed hematopoietic stem cell‑negative markers CD34 (0.21%), CD45 (0.45%) and CD11 (1.08%) as determined by flow cytometry. Trilineage differentiation assays confirmed that NPSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials, consistent with the phenotypic characteristics of mesenchymal stem cells. In vitro spheroid culture induced NPSCs to form regular spheroids with smooth edges, with an average diameter of 52.3±11.6 μm. Cell cycle analysis revealed that the proportion of G0‑phase cells in the SP group was significantly higher than that in the ML group (P<0.05). Moreover, EdU‑positive cell proportion was markedly lower in the SP group than in the ML group (P<0.05), indicating a typical quiescent state. RT-qPCR showed that the expression of P27, SOX2 and CD34 in the SP group were significantly higher than those in the ML group (P<0.05). Bulk RNA‑sequencing demonstrated significant differentially expressed genes between the SP and ML groups, with significant enrichment of the autophagy pathway and the PI3K‑Akt‑mTOR pathway. Western blotting demonstrated that, compared with the ML group, the ratio of LC3‑Ⅱ/LC3‑Ⅰ was significantly increased, while the expression of p62 was notably decreased in the SP group. The expression levels of key molecules in the PI3K-Akt-mTOR pathway, such as PI3K, p‑Akt and p‑mTOR were all significantly downregulated (P<0.05). TEM revealed a markedly higher number of autolysosomes in the SP group than in the ML group. Rescue experiments further verified the regulatory relationship between the PI3K‑Akt‑mTOR pathway and autophagy (P<0.05). Clinical sample detection showed that the expression levels of Ki67 and p‑mTOR in nucleus pulposus tissues were significantly higher in juvenile patients than in adult patients, whereas the content of LC3B was significantly lower (P<0.05).

Conclusion

In vitro 3D spheroid culture can effectively induce NPSCs to enter a quiescent state and maintain their stemness. The underlying mechanism may involve the activation of autophagy via inhibition of the PI3K‑Akt‑mTOR pathway.

, authors=Tong ZHU, Jiabin LIU, Runzhi ZHAO, Zhuolin YANG, Bo HUANG, authorsList=Tong ZHU, Jiabin LIU, Runzhi ZHAO, Zhuolin YANG, Bo HUANG, authorCompany=null, correspAuthors=Bo HUANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=© 2026 Journal of Army Medical University, copyrightOwner=Editorial Office of Journal of Army Medical University, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1320442413810832071, articleId=1320442409910129339, tenantId=1146029695717560320, journalId=1319373162502258839, language=CN, title=成团培养髓核干细胞呈静息状态并伴有PI3K-Akt-mTOR 通路受抑和自噬激活, columnId=1320434418343329969, journalTitle=陆军军医大学学报, columnName=论著·基础医学, runingTitle=null, highlight=null, articleAbstract=
目的

椎间盘退变（intervertebral disc degeneration，IVDD）是下腰痛的主要病因，髓核干细胞（nucleus pulposus stem cells ， NPSCs）干性耗竭为其关键机制，维持其静息状态对延缓退变具有重要意义。旨在探讨体外成团培养对NPSCs静息状态的诱导作用及其调控机制，为IVDD的靶向干预提供实验依据。

方法

收集陆军军医大学第二附属医院骨科2025年12月至2026年1月进行腰椎手术的未成年患者和成年患者的椎间盘组织，进行组织切片并通过HE染色观察形态差异。提取24只8周龄SD雄性大鼠［体质量（350±50）g］的NPSCs。通过流式细胞术检测干细胞免疫表型，三系分化实验验证NPSCs的干性。正常平面培养的NPSCs作为单层培养（monolayer culture，ML）组；利用三维球形培养法对NPSCs建立成团培养模型，作为成团培养（sphere culture，SP）组。显微镜观察细胞形态，流式细胞术进行周期分析，EdU染色鉴定其静息状态。对正常平面培养和成团培养的NPSCs进行bulk转录组测序和分析。使用RT-qPCR、Western blotting和透射电镜等方法，检测自噬相关分子及PI3K-Akt-mTOR通路关键分子的表达和自噬溶酶体结构。

结果

临床组织切片检测显示，未成年患者髓核细胞呈散点样分布，成年患者髓核细胞呈成团簇状分布，且成年患者髓核细胞成团分布占比显著高于未成年患者（P<0.01）。成功分离的大鼠NPSCs经流式细胞术检测，显示高表达间充质干细胞阳性标志物CD90（99.4%）、CD44（99.8%）、CD73（94.2%），低表达造血干细胞阴性标志物CD34（0.21%）、CD45（0.45%）、CD11（1.08%）。三系分化实验证实其具有成骨、成软骨、成脂分化潜能，符合间充质干细胞表型特征。体外成团培养可诱导NPSCs聚集形成球形度规整、边缘光滑的球形细胞团，平均直径为（52.3±11.6）μm；细胞周期检测显示，SP组G0期细胞比例显著高于ML组（P<0.05），且SP组EdU阳性细胞比例均较ML组显著降低（P<0.05），呈典型静息状态。RT-qPCR检测显示，SP组的P27、SOX2和CD34表达显著高于ML组（P<0.05）。Bulk转录组测序结果显示，SP组与ML组间存在显著差异表达基因，且自噬和PI3K-Akt-mTOR通路呈显著富集趋势。Western blotting检测结果显示，与ML组相比，SP组成团细胞自噬相关分子LC3-Ⅱ/LC3-Ⅰ比值显著升高、P62表达显著降低，PI3K-Akt-mTOR通路关键分子PI3K、p-Akt、p-mTOR表达均显著下调（P<0.05）；透射电镜下可见SP组细胞内自噬溶酶体数量较ML组显著增加；挽救实验进一步验证PI3K-Akt-mTOR通路与自噬的调控关联（P<0.05）。临床样本检测显示，未成年患者髓核组织Ki67、p-mTOR表达水平显著高于成年患者，而LC3B含量显著低于成年患者（P<0.05）。

结论

体外成团培养可有效诱导NPSCs进入静息状态并维持干性，其机制可能通过抑制PI3K-Akt-mTOR通路激活自噬来实现。

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黄博，E-mail：
HUANG Bo，E-mail：
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Nature， 2016， 529（7584）： 37-42., articleTitle=Autophagy maintains stemness by preventing senescence, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1320442414209290952, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, xref=1, ext=[AuthorCompanyExt(id=1320442414213485257, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, companyId=1320442414209290952, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1Department of Orthopedics， Second Affiliated Hospital， Army Medical University （Third Military Medical University）， Chongqing， China), AuthorCompanyExt(id=1320442414221873866, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, companyId=1320442414209290952, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1陆军军医大学（第三军医大学）第二附属医院骨科，重庆)])], figs=[ArticleFig(id=1320442419678663409, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=EN, label=Figure 1, caption=Distribution characteristics of human nucleus pulposus tissues and identification of rat NPSCs, figureFileSmall=tFW3gifAU1+EX6gdiBnd5w==, figureFileBig=ni/pIFwm5JUaQlFC3b+0cQ==, tableContent=null), ArticleFig(id=1320442419749966578, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=CN, label=图1, caption=人体髓核细胞分布特征及大鼠NPSCs的鉴定

A and B： Representative images of HE staining of juvenile （A） and adult （B） nucleus pulposus tissues； C： Representative images of microscopic observation of tissue block-cultured rat NPSCs after monolayer culture； D： Surface markers of rat NPSCs by flow cytometry； E： Tri-lineage differentiation capacity of rat NPSCs

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A： Morphology of rat NPSCs in SP group after spheroid culture； B： Morphology of rat NPSCs in Re-ML group after re-plating； C： Senescence analysis in the ML and SP groups； D： Cell cycle distribution and G0-phase proportion in the ML and SP groups； E： EdU proliferation assay in the ML and SP groups； F： Expression levels of P27， SOX2， CD34 in the ML and SP groups by RT-qPCR； G： Representative Western blotting images of P27 and SOX2 in the ML and SP groups； H： Representative Western blotting images of OCT4 and Nanog in the ML， SP and Re-ML groups

, figureFileSmall=QzjaJfvRrBo8W+0r5Frm7w==, figureFileBig=sPJzdwabv6lGzh9nLjoffA==, tableContent=null), ArticleFig(id=1320442422040056565, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=EN, label=Figure 3, caption=RNA-seq results in NPSCs after spheroid culture, figureFileSmall=rV2mYML6z7aOOipstrwKew==, figureFileBig=mfPjw2ZwP6R0WQroFvscQg==, tableContent=null), ArticleFig(id=1320442422115554038, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=CN, label=图3, caption=NPSCs成团培养后的测序结果

A to C： Transcriptomic differential expression analysis between the ML and SP groups； D and E： Top 30 GO functional and KEGG pathway enrichment analyses of differentially expressed genes between the ML group and SP group； F： KEGG enrichment analysis of genes associated with the PI3K-Akt pathway and autophagy； G and H： Expression changes and RT-qPCR validation of differentially expressed genes related to the PI3K-Akt pathway and autophagy

, figureFileSmall=rV2mYML6z7aOOipstrwKew==, figureFileBig=mfPjw2ZwP6R0WQroFvscQg==, tableContent=null), ArticleFig(id=1320442422178468599, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=EN, label=Figure 4, caption=Detection of PI3K-Akt-mTOR pathway and autophagy-related indicators in rat NPSCs after spheroid culture, figureFileSmall=CkbwY+P9woX2eJNF1moHrw==, figureFileBig=BwAsu5wykQVBhzfQfsDSEw==, tableContent=null), ArticleFig(id=1320442422249771768, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=CN, label=图4, caption=大鼠NPSCs成团培养后PI3K-Akt-mTOR通路及自噬相关指标的检测

A to C： Protein expression of the PI3K-Akt-mTOR pathway and autophagy in the ML and SP groups； D： Immunofluorescence assay of Ki67， LC3B， and p-mTOR in juvenile and adult human nucleus pulposus tissues； E： Transmission electron microscopy of autophagy-related ultrastructures in the ML and SP groups

, figureFileSmall=CkbwY+P9woX2eJNF1moHrw==, figureFileBig=BwAsu5wykQVBhzfQfsDSEw==, tableContent=null), ArticleFig(id=1320442422308492025, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=EN, label=Figure 5, caption=Regulatory effect of PI3K-Akt-mTOR and autophagy on spheroid culture-induced quiescent state of NPSCs, figureFileSmall=JRtJ4cx5mSff3pvYQubl8A==, figureFileBig=d5+LTzkbS300lSOC7+m7jg==, tableContent=null), ArticleFig(id=1320442422367212282, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=CN, label=图5, caption=PI3K-Akt-mTOR和自噬通路对成团培养诱导NPSCs静息状态的调控作用检测

A and B： Protein expression of autophagy- and mTOR-related markers in NPSCs after CQ or MHY1485 treatment； C： Cell cycle distribution and G0-phase proportion after pharmacological interventions

, figureFileSmall=JRtJ4cx5mSff3pvYQubl8A==, figureFileBig=d5+LTzkbS300lSOC7+m7jg==, tableContent=null), ArticleFig(id=1320442422451098363, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=EN, label=Table 1, caption=

Primer sequences for RT-qPCR

, figureFileSmall=null, figureFileBig=null, tableContent=
GenesSequences（5'→3'）
GAPDHForward：GCCAGCCTCGTCTCATAGAC
Reverse：AGTGATGGCATGGACTGTGG
P27Forward：GCTTTCGGGG TGTTTTTCCC
Reverse：TCTTCATGATTGACCGGGCC
SOX2Forward：GATCAGCATGTACCTCCCCG
Reverse：CATCGCCCGGAGTCTAGTTC
CD34Forward：AGCTGGGGATCCAAAGCTTC
Reverse：GTGCAGAGAGAGGGCAGTTT
Pik3cdForward：GATGGACTCAAACGGCAGGA
Reverse：ACTCGTTCATGGTCACACGG
Akt1Forward：CTGCACTTGTTGTTGCCTCC
Reverse：ATCTCCCTGGCTGCTCCTTA
MtorForward：CTGCACTTGTTGTTGCCTCC
Reverse：ATCTCCCTGGCTGCTCCTTA
Atg2aForward：CCTGCGGCGAGATTATGTCT
Reverse：GCAGAACTCGTCACTGTCCA
Atg4cForward：CATCAGCAT TCCCACCCAGT
Reverse：AAAACTCACCACAGTGCCCA
Atg13Forward：AGCCCGGAGTCACTCTATGT
Reverse：CACGAACTGTCTGGAAGCCT
), ArticleFig(id=1320442422522401532, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442409910129339, language=CN, label=表1, caption=

RT-qPCR引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
GenesSequences（5'→3'）
GAPDHForward：GCCAGCCTCGTCTCATAGAC
Reverse：AGTGATGGCATGGACTGTGG
P27Forward：GCTTTCGGGG TGTTTTTCCC
Reverse：TCTTCATGATTGACCGGGCC
SOX2Forward：GATCAGCATGTACCTCCCCG
Reverse：CATCGCCCGGAGTCTAGTTC
CD34Forward：AGCTGGGGATCCAAAGCTTC
Reverse：GTGCAGAGAGAGGGCAGTTT
Pik3cdForward：GATGGACTCAAACGGCAGGA
Reverse：ACTCGTTCATGGTCACACGG
Akt1Forward：CTGCACTTGTTGTTGCCTCC
Reverse：ATCTCCCTGGCTGCTCCTTA
MtorForward：CTGCACTTGTTGTTGCCTCC
Reverse：ATCTCCCTGGCTGCTCCTTA
Atg2aForward：CCTGCGGCGAGATTATGTCT
Reverse：GCAGAACTCGTCACTGTCCA
Atg4cForward：CATCAGCAT TCCCACCCAGT
Reverse：AAAACTCACCACAGTGCCCA
Atg13Forward：AGCCCGGAGTCACTCTATGT
Reverse：CACGAACTGTCTGGAAGCCT
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朱桐 , 刘嘉斌 , 赵润智 , 杨卓霖 , 黄博
陆军军医大学学报 | 论著·基础医学 2026,48(9): 1181-1193
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陆军军医大学学报 |论著·基础医学 2026 , 48 (9) : 1181 -1193
成团培养髓核干细胞呈静息状态并伴有PI3K-Akt-mTOR 通路受抑和自噬激活
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朱桐, 刘嘉斌, 赵润智, 杨卓霖, 黄博
作者信息
  • 1陆军军医大学（第三军医大学）第二附属医院骨科，重庆
通讯作者:
黄博，E-mail：
HUANG Bo，E-mail：
Spheroid culture induces a quiescent state in nucleus pulposus stem cells by inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy
Tong ZHU, Jiabin LIU, Runzhi ZHAO, Zhuolin YANG, Bo HUANG
Affiliations
  • 1Department of Orthopedics， Second Affiliated Hospital， Army Medical University （Third Military Medical University）， Chongqing， China
出版时间: 2026-05-15 doi: 10.16016/j.2097-0927.202601032
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摘要
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目的

椎间盘退变（intervertebral disc degeneration，IVDD）是下腰痛的主要病因，髓核干细胞（nucleus pulposus stem cells ， NPSCs）干性耗竭为其关键机制，维持其静息状态对延缓退变具有重要意义。旨在探讨体外成团培养对NPSCs静息状态的诱导作用及其调控机制，为IVDD的靶向干预提供实验依据。

方法

收集陆军军医大学第二附属医院骨科2025年12月至2026年1月进行腰椎手术的未成年患者和成年患者的椎间盘组织，进行组织切片并通过HE染色观察形态差异。提取24只8周龄SD雄性大鼠［体质量（350±50）g］的NPSCs。通过流式细胞术检测干细胞免疫表型，三系分化实验验证NPSCs的干性。正常平面培养的NPSCs作为单层培养（monolayer culture，ML）组；利用三维球形培养法对NPSCs建立成团培养模型，作为成团培养（sphere culture，SP）组。显微镜观察细胞形态，流式细胞术进行周期分析，EdU染色鉴定其静息状态。对正常平面培养和成团培养的NPSCs进行bulk转录组测序和分析。使用RT-qPCR、Western blotting和透射电镜等方法，检测自噬相关分子及PI3K-Akt-mTOR通路关键分子的表达和自噬溶酶体结构。

结果

临床组织切片检测显示，未成年患者髓核细胞呈散点样分布，成年患者髓核细胞呈成团簇状分布，且成年患者髓核细胞成团分布占比显著高于未成年患者（P<0.01）。成功分离的大鼠NPSCs经流式细胞术检测，显示高表达间充质干细胞阳性标志物CD90（99.4%）、CD44（99.8%）、CD73（94.2%），低表达造血干细胞阴性标志物CD34（0.21%）、CD45（0.45%）、CD11（1.08%）。三系分化实验证实其具有成骨、成软骨、成脂分化潜能，符合间充质干细胞表型特征。体外成团培养可诱导NPSCs聚集形成球形度规整、边缘光滑的球形细胞团，平均直径为（52.3±11.6）μm；细胞周期检测显示，SP组G0期细胞比例显著高于ML组（P<0.05），且SP组EdU阳性细胞比例均较ML组显著降低（P<0.05），呈典型静息状态。RT-qPCR检测显示，SP组的P27、SOX2和CD34表达显著高于ML组（P<0.05）。Bulk转录组测序结果显示，SP组与ML组间存在显著差异表达基因，且自噬和PI3K-Akt-mTOR通路呈显著富集趋势。Western blotting检测结果显示，与ML组相比，SP组成团细胞自噬相关分子LC3-Ⅱ/LC3-Ⅰ比值显著升高、P62表达显著降低，PI3K-Akt-mTOR通路关键分子PI3K、p-Akt、p-mTOR表达均显著下调（P<0.05）；透射电镜下可见SP组细胞内自噬溶酶体数量较ML组显著增加；挽救实验进一步验证PI3K-Akt-mTOR通路与自噬的调控关联（P<0.05）。临床样本检测显示，未成年患者髓核组织Ki67、p-mTOR表达水平显著高于成年患者，而LC3B含量显著低于成年患者（P<0.05）。

结论

体外成团培养可有效诱导NPSCs进入静息状态并维持干性，其机制可能通过抑制PI3K-Akt-mTOR通路激活自噬来实现。

髓核干细胞  /  成团培养  /  静息状态  /  自噬  /  PI3K-Akt-mTOR通路
Objective

Intervertebral disc degeneration (IVDD) is a primary cause of low back pain, and stemness exhaustion of nucleus pulposus stem cells (NPSCs) represents a key underlying mechanism. Thus, maintaining the quiescent state of NPSCs is of great significance in delaying disc degeneration. This study aimed to investigate the inductive effect of in vitro three‑dimensional (3D) spheroid culture on the quiescent state of NPSCs and its regulatory mechanism, thereby providing experimental evidence for targeted intervention in IVDD.

Methods

Intervertebral disc tissues were collected from juvenile and adult patients undergoing lumbar surgery in our department between December 2025 and January 2026. Tissue sections were prepared, and morphological differences were observed through HE staining. NPSCs were isolated from 24 male SD rats (8 weeks old, weighing 350±50 g). Stem cell immunophenotypes were identified by flow cytometry, and the stemness of NPSCs was verified by trilineage differentiation assay. NPSCs cultured under routine 2D conditions served as the monolayer culture (ML) group, while NPSCs cultured using the 3D spheroid culture system were established as the spheroid culture (SP) group. Cell morphology was observed under a microscope, cell cycle distribution was analyzed by flow cytometry, and the quiescent state was identified by EdU staining. Bulk transcriptome sequencing and bioinformatic analysis were performed on NPSCs from the ML and SP groups. The expression of autophagy‑related molecules and key molecules in the PI3K‑Akt‑mTOR signaling pathway, as well as the ultrastructure of autolysosomes, were detected by RT‑qPCR, Western blotting and transmission electron microscopy (TEM), respectively.

Results

Clinical histological observation showed that nucleus pulposus cells in juvenile patients were scattered in distribution, whereas those in adult patients exhibited clustered distribution, and the proportion of clustered nucleus pulposus cells was significantly higher in adult patients than in juvenile patients(P<0.01). The successfully isolated rat NPSCs highly expressed mesenchymal stem cell‑positive markers CD90 (99.4%), CD44 (99.8%) and CD73 (94.2%), and lowly expressed hematopoietic stem cell‑negative markers CD34 (0.21%), CD45 (0.45%) and CD11 (1.08%) as determined by flow cytometry. Trilineage differentiation assays confirmed that NPSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials, consistent with the phenotypic characteristics of mesenchymal stem cells. In vitro spheroid culture induced NPSCs to form regular spheroids with smooth edges, with an average diameter of 52.3±11.6 μm. Cell cycle analysis revealed that the proportion of G0‑phase cells in the SP group was significantly higher than that in the ML group (P<0.05). Moreover, EdU‑positive cell proportion was markedly lower in the SP group than in the ML group (P<0.05), indicating a typical quiescent state. RT-qPCR showed that the expression of P27, SOX2 and CD34 in the SP group were significantly higher than those in the ML group (P<0.05). Bulk RNA‑sequencing demonstrated significant differentially expressed genes between the SP and ML groups, with significant enrichment of the autophagy pathway and the PI3K‑Akt‑mTOR pathway. Western blotting demonstrated that, compared with the ML group, the ratio of LC3‑Ⅱ/LC3‑Ⅰ was significantly increased, while the expression of p62 was notably decreased in the SP group. The expression levels of key molecules in the PI3K-Akt-mTOR pathway, such as PI3K, p‑Akt and p‑mTOR were all significantly downregulated (P<0.05). TEM revealed a markedly higher number of autolysosomes in the SP group than in the ML group. Rescue experiments further verified the regulatory relationship between the PI3K‑Akt‑mTOR pathway and autophagy (P<0.05). Clinical sample detection showed that the expression levels of Ki67 and p‑mTOR in nucleus pulposus tissues were significantly higher in juvenile patients than in adult patients, whereas the content of LC3B was significantly lower (P<0.05).

Conclusion

In vitro 3D spheroid culture can effectively induce NPSCs to enter a quiescent state and maintain their stemness. The underlying mechanism may involve the activation of autophagy via inhibition of the PI3K‑Akt‑mTOR pathway.

nucleus pulposus stem cells  /  spheroid culture  /  quiescent state  /  autophagy  /  PI3K-Akt-mTOR pathway
朱桐, 刘嘉斌, 赵润智, 杨卓霖, 黄博. 成团培养髓核干细胞呈静息状态并伴有PI3K-Akt-mTOR 通路受抑和自噬激活. 陆军军医大学学报, 2026 , 48 (9) : 1181 -1193 . DOI: 10.16016/j.2097-0927.202601032
Tong ZHU, Jiabin LIU, Runzhi ZHAO, Zhuolin YANG, Bo HUANG. Spheroid culture induces a quiescent state in nucleus pulposus stem cells by inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy[J]. Journal of Army Medical University, 2026 , 48 (9) : 1181 -1193 . DOI: 10.16016/j.2097-0927.202601032
正文
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椎间盘退变（intervertebral disc degeneration， IVDD）是下腰痛的首要病理诱因，其发病率居高不下，不仅严重降低全球4亿多患者的生活质量，更造成重大的全球性公共卫生负担［1-3］。椎间盘作为脊柱力学功能的核心承载单元，主要由髓核、纤维环及软骨终板3部分构成，其内环境稳态的失衡是驱动IVDD发生发展的关键病理环节［4］。其中，髓核组织的核心功能主要由髓核干细胞（nucleus pulposus stem cells，NPSCs）来维持，而NPSCs可以通过持续分泌蛋白聚糖来调控细胞外基质的稳态，这些对维持椎间盘正常生理功能至关重要［5］。NPSCs作为髓核细胞的前体细胞［6］，系统开展NPSCs的形态学特征及功能调控机制研究，对于阐明IVDD病理进程、开发靶向干预策略具有重要的理论与临床意义。
静息状态是干细胞群体中普遍存在的一种生理状态，特指细胞主动退出细胞周期并进入可逆性周期阻滞的状态，此状态下干细胞可有效维持自身稳态并具有自我更新能力［7］。处于静息状态的干细胞，其基础代谢水平、生物合成活性及能量需求均显著下调，这一适应性特征使其能够耐受外界可能会面对的营养匮乏、低氧等恶劣微环境并长期存活［8-9］。与细胞衰老的不可逆性不同，静息状态的干细胞在特定刺激信号激活下可重新进入细胞周期，启动增殖与分化程序［10］。已有研究证实，椎间盘中存在一群髓核祖细胞，该群体以慢周期状态或者静息状态存在，在椎间盘损伤后可被激活，参与组织修复与再生过程［11-13］。然而，目前关于NPSCs静息状态的调控规律及其分子机制尚未完全阐明，相关研究仍处于探索阶段。
三维成团培养模型作为模拟体内细胞天然微环境的经典体外培养模型，已被大量研究证实可精准调控多种干细胞的增殖活性及静息状态［14-16］。同时，现有研究已证实自噬通路的激活与干细胞静息状态的建立及维持密切相关［17］。然而，截至目前，成团培养介导的NPSCs是否可以进入静息状态、成团后的NPSCs在维持IVDD进程中的作用、与自噬通路之间的调控关联尚未明确。基于上述研究的缺乏，本研究旨在系统探讨成团培养技术维持NPSCs静息状态的效应及潜在自噬相关分子机制，以期为IVDD的预防与靶向治疗提供部分理论依据与技术思路。
1　材料与方法
收起
1.1　材料
1.1.1　标本的获取
人的髓核组织来自2025年12月至2026年1月在陆军军医大学第二附属医院骨科接受腰椎手术患者。按年龄分为未成年组（年龄<18岁）和成年组（年龄≥18岁）。未成年组的髓核组织取自4例脊柱骨折或畸形患者，年龄13~17岁；成年组的髓核组织取自5例IVDD患者，年龄45~70岁。本研究经陆军军医大学第二附属医院医学伦理委员会批准（2025-研第455-01）。
1.1.2　实验试剂
Ⅱ型胶原酶购自北京Solarbio公司；Dulbecco改良Eagle培养基/营养混合物F-12（DMEM/F12）培养基、青霉素-链霉素购自德阳Sperikon公司；FBS、总RNA提取试剂盒购自武汉ServiceBio公司；ABScript 3 RT Master Mix逆转录试剂盒、ECL超敏化学发光试剂盒购自武汉ABclonal公司；SYBR Prime qPCR试剂盒（Fast HS）购自重庆Bioground公司；OriCell®大鼠间充质干细胞表面标志物检测试剂盒，OriCell®大鼠骨髓间充质干细胞成脂、成骨、成软骨分化试剂盒购自美国OriCell公司；PI/RNase染色缓冲液购自美国BD Pharmingen公司；Ki67（D3B5）兔单克隆抗体（Alexa Fluor® 488偶联）购自美国Cell Signaling Technology公司；Senescence-Tracker™衰老细胞近红外荧光探针（C0603）、EdU细胞增殖试剂盒（AF647标记）购自江苏碧云天生物技术有限公司；GAPDH抗体、P62抗体、Anti-Rabbit抗体、Anti-Mouse抗体购自武汉Abclonal公司；P27抗体、SOX2抗体、LC3B抗体、p-mTOR抗体、p-Akt抗体、mTOR抗体、PI3K抗体、Akt抗体购自浙江华安公司；OCT4抗体和Nanog抗体购自北京Bioss公司。
1.2　方法
1.2.1　大鼠NPSCs的分离与培养
取24只8周龄SD雄性大鼠［体质量（350±50）g］，用2%戊巴比妥钠麻醉后处死。大鼠由陆军军医大学第二附属医院实验动物中心提供， NPSCs分离自大鼠椎间盘组织。本研究所有动物实验经陆军军医大学实验动物福利伦理委员会审批（AMUWEC20255372），实验操作符合动物伦理规范。大鼠饲养于陆军军医大学第二附属医院实验动物中心SPF级动物房，环境温度22~25 ℃、相对湿度50%~60%，保持12 h光照/12 h黑暗的节律，自由摄食饮水，笼具每周清洁更换2次。实验由专业人员操作，全程无菌分离组织，最大程度减少动物痛苦；实验期间每日监测大鼠状态，设定体质量下降超20%、精神萎靡、活动障碍等为人道终点，本研究未出现预期及意外不良事件，无触发人道终点情况。
取鼠尾浸泡在碘伏中30 min后，在无菌条件下剥离皮肤和肌肉，显露椎间盘，挤压同时使用无菌刀片切割椎间盘，髓核组织呈白色胶冻样即游离至刀片侧，加入0.25%Ⅱ型胶原酶，于37 ℃消化30 min。将细胞悬液离心，室温以250×g离心3 min，弃去上清液。NPSCs采用DMEM/F12培养基培养，培养基中添加10%FBS和1%青霉素-链霉素，培养环境为37 ℃、5%CO2湿润培养箱。当细胞融合度达到95%时，用0.25%胰蛋白酶消化贴壁细胞，按细胞悬液体积的1∶3比例传代。实验选用第三代（P3）NPSCs进行后续研究，贴壁培养的NPSCs命名为单层培养（monolayer culture，ML）组。
1.2.2.1　微孔板三维培养法构建大鼠NPSCs球状体
根据参考文献［18］，当第三代NPSCs体外培养至80%融合度时，经胰蛋白酶消化后离心。采用微孔板（Aggrewell™，美国）三维球状体培养法构建NPSCs球状体：在24孔微孔板中通过离心形成球状体，通过接种适宜细胞密度（1×106个/孔）控制球状体大小。每日更换50%~75%的完全DMEM/F12培养基。培养3 d后，收集NPSCs球状体用于后续实验，收集的NPSCs球状体命名为成团培养（sphere culture， SP）组；若需获得单细胞，可将球状体经胰蛋白酶消化为单细胞悬液。将球状体NPSCs重新置于普通培养瓶中继续贴壁生长的NPSCs组命名为重新铺板（Re-ML）组。
1.2.2.2　倒置相差显微镜观察及检测大鼠NPSCs球状体形态和直径
采用倒置相差显微镜（Olympus，日本）观察成团形态，以球形度规整、边缘光滑、无明显分散细胞为形态合格标准；随机选取3个视野，每个视野随机选取12个细胞团，使用Image J软件测量细胞团最大直径，统计直径集中于100~200 μm。
1.2.3　HE染色检测人髓核组织形态结构
将4%多聚甲醛固定后的髓核组织样品经梯度脱水、二甲苯透明处理后，进行石蜡包埋，制作2 μm连续切片。切片经二甲苯脱蜡后进行染色。苏木素和伊红染液（ServiceBio，武汉）染色5~10 min，自来水冲洗10 min返蓝；1%盐酸乙醇分化3~5 s，自来水冲洗5 min。伊红染液染色30~60 s，自来水冲洗10 s。使用中性树胶封片。置于光学显微镜下观察组织形态结构，拍摄图像并进行形态学分析。
1.2.4　流式细胞术检测大鼠NPSCs表面标志物表达
采用OriCell®大鼠间充质干细胞表面标志物检测试剂盒检测第3代NPSCs的表面标志物表达。该试剂盒包含阳性标志物（CD44、CD90、CD73）一抗、阴性标志物（CD34、CD45）一抗、同型对照抗体及藻红蛋白（PE）标记二抗。简要实验流程：将NPSCs重悬于流式细胞术缓冲液（含0.1%FBS的1×磷酸盐缓冲液）中，调整细胞浓度至3×106/mL。在标记好的1.5 mL离心管中，加入100 μL细胞悬液（约3×105个细胞）和2 μL一抗（或同型对照抗体），于4 ℃孵育30 min。用200 μL缓冲液洗涤2次（250×g，5 min）后，加入2 μL二抗，继续于4 ℃孵育30 min。再次洗涤细胞后，将其重悬于300~500 μL缓冲液中，立即采用流式细胞仪（Beckman Coulter，美国）检测。使用FlowJo软件进行数据分析。
1.2.5　油红O染色检测大鼠NPSCs成脂分化能力
采用OriCell®大鼠骨髓间充质干细胞成脂分化试剂盒诱导NPSCs成脂分化。简要实验流程：将第三代NPSCs以2×104个/cm²的密度接种于培养皿中，置于37 ℃、5%CO2细胞培养箱中培养至融合度100%。按说明书配制好成脂诱导分化培养基，成脂诱导阶段，交替使用成脂诱导培养基A液和B液培养细胞，A液3 d，B液1 d，期间每日观察细胞状态。约3周后，分化完成后，用10%福尔马林固定细胞30 min，随后加入油红O工作液染色30 min。经磷酸盐缓冲液洗涤后，在显微镜下观察细胞形态。
1.2.6　阿尔辛蓝染色检测大鼠NPSCs成软骨分化能力
采用OriCell®大鼠骨髓间充质干细胞成软骨分化试剂盒诱导NPSCs成软骨分化。收集4×105个第三代NPSCs，经250×g离心4 min后，用磷酸盐缓冲液洗涤。成软骨诱导完全培养基的配制方法如下：在每1毫升预混液（含添加剂Ⅰ的基础培养基）中加入10 μL成软骨分化添加剂Ⅱ。将细胞重悬于0.5 mL完全培养基中，转移至15 mL离心管（盖子松动），置于37 ℃、5%CO2环境中培养。每2~3天更换0.5 mL新鲜完全培养基，约2周后，管内形成直径1.5~2.0 mm的软骨球。将软骨球用10%福尔马林固定30 min以上，随后进行脱水、石蜡包埋，切片厚度为3 μm。脱蜡脱水后，将切片于阿尔辛蓝染色1 h，洗涤后在显微镜下观察。
1.2.7　茜素红S染色检测大鼠NPSCs成骨分化能力
采用OriCell®大鼠骨髓间充质干细胞成骨分化试剂盒诱导NPSCs成骨分化。按说明书配制好成骨诱导分化培养基，将第三代NPSCs接种于培养皿中，加入成骨诱导培养基，置于37 ℃、5%CO2培养箱中培养，每3天更换1次培养基。约2周后，细胞分化成功后（以矿化结节形成为标志），用4%多聚甲醛固定细胞30 min，洗涤后，加入茜素红S溶液室温染色15~20 min。洗涤后在显微镜下观察细胞形态。
1.2.8　衰老细胞近红外荧光探针及流式细胞术检测各组大鼠NPSCs衰老状态
采用衰老细胞近红外荧光探针（C0603）检测ML组和SP组NPSCs的衰老状态。先取10 mmol/L探针储存液按1∶1 000的体积比加入培养液，对贴壁细胞的成团细胞过夜孵育，孵育后用胰蛋白酶消化贴壁细胞并收集，用胰蛋白酶将球状体消化为单细胞悬液，2组样本通过流式细胞仪检测荧光阳性细胞比例，以未处理的年轻NPSCs为阴性对照设定荧光阈值，计算衰老细胞比例。
1.2.9　PI/RNase染色及流式细胞术检测各组大鼠NPSCs细胞周期
采用PI/RNase染色缓冲液和Ki67（D3B5）兔单克隆抗体（Alexa Fluor® 488偶联）分析细胞周期。将2组细胞经胰蛋白酶消化后，收集约1×106个细胞。用磷酸盐缓冲液洗涤后，加入预冷的70%乙醇于4 ℃固定12 h，随后用磷酸盐缓冲液洗涤2次。加入Ki67（D3B5）兔单克隆抗体，室温避光孵育1 h，再加入PI染色液室温避光孵育20 min。采用流式细胞仪获取数据，使用FlowJo软件进行数据分析。
1.2.10　EdU细胞增殖试剂盒及共聚焦显微镜检测各组大鼠NPSCs增殖能力
采用EdU细胞增殖试剂盒（AF647标记）对2组细胞进行EdU实验。将细胞置于含10 μmol/L EdU的完全培养基中孵育4 h（细胞周期1/10），用4%多聚甲醛（PFA）固定15 min，加入0.3%曲拉通X-100通透15 min。按照试剂盒说明书，采用Click-iT EdU Alexa Fluor 555检测系统检测孵育的EdU。采用共聚焦显微镜（Olympus公司，日本）拍摄图像。使用Image J软件计数DAPI阳性细胞核（代表总细胞数）和EdU阳性细胞核（代表增殖细胞数）。增殖率计算公式：增殖率=（EdU⁺细胞核数/DAPI⁺细胞核数）×100%。
1.2.11　Bulk RNA-Seq分析各组大鼠NPSCs基因表达谱
本研究设置2组进行bulk转录组测序，即ML组和SP组。各组处理完成后，采用TRIzol法提取大鼠NPSCs总RNA并送检。RNA-Seq流程（包括样本评估、文库构建、质量控制和测序）由杭州广科安德生物技术有限公司完成。采用Illumina NovaSeq 6000测序平台进行RNA-Seq，对细胞在特定时间段内表达的所有mRNA转录本进行测序。文库构建采用Illumina TruSeq™ RNA样本制备试剂盒。测序获得的原始数据中包含少量带测序接头或低质量的序列。为保证数据分析的质量和可靠性，采用FAST软件对原始数据进行过滤，过滤标准如下：①去除含接头的序列；②去除含模糊碱基（以N表示）的序列；③去除低质量序列（Qphred≤20的碱基占总碱基的50%及以上）。本研究以|Log2FC|≥1且校正后P值（P adj）<0.05为标准筛选DEGs。采用DESeq2软件进行差异表达分析，采用Cluster Profiler软件对差异表达基因进行GO功能富集和KEGG通路富集分析。
1.2.12　RT-qPCR检测各组大鼠NPSCs中P27、SOX2、CD34等基因表达水平
使用总RNA提取试剂盒提取细胞总RNA，通过分光光度法测定总RNA纯度。随后，采用含基因组DNA去除剂的ABScript 3 RT Master Mix逆转录试剂盒将RNA逆转录为互补DNA（cDNA）。采用SYBR Prime qPCR试剂盒（Fast HS）进行定量PCR检测。以GAPDH为内参基因，采用2-△△Ct法计算目的基因的相对表达量。实验数据为3次独立实验获得的平均值。目的基因的引物序列见表1。
1.2.13　Western blotting检测各组大鼠NPSCs中P27、SOX2等相关蛋白表达水平
大鼠平面培养和成团培养的NPSCs在RIPA缓冲液中进行裂解。用BCA蛋白法测定蛋白质含量。通过SDS-PAGE对蛋白质进行分离，然后转移到PVDF膜上。取出PVDF膜放至5%脱脂奶粉中封闭1 h。一抗GAPDH（1∶50 000）、P27（1∶2 000）、SOX2（1∶1 000）、LC3B（1∶1 000）、p-mTOR（1∶1 000）、PI3K（1∶1 000）、OCT4（1∶1 000）、Nanog（1∶1 000）、P62（1∶10 000）、p-Akt（1∶5 000）、mTOR（1∶5 000）、Akt（1∶5 000）。将PVDF膜与一抗在4 ℃下孵育过夜（10~12 h），将膜与相对应的二抗孵育，二抗Anti-Rabbit（1∶10 000）、Anti-Mouse（1∶10 000）。采用ECL超敏化学发光试剂盒（A液∶B液=1∶1）显影，ChemiDoc XRS+凝胶成像系统采集图像，Image J软件分析蛋白条带灰度值，以GAPDH为内参计算目的蛋白相对表达量。
1.2.14　透射电子显微镜观察各组大鼠NPSCs超微结构
取ML组和SP组的NPSCs，用2.5%戊二醛固定液（ServiceBio公司，武汉）4 ℃固定2.5 h。而后外送至百奥斯生物公司，使用1%锇酸室温后固定 1 h，梯度乙醇（30%、50%、70%、80%、90%、100%）脱水，环氧树脂618包埋；经超薄切片机制作70 nm超薄切片，采用醋酸铀-柠檬酸铅双染法室温染色15 min；日立H-7650透射电镜下观察细胞超微结构，重点观察溶酶体、自噬溶酶体等结构变化，并拍摄代表性图像。
1.2.15　药物干预结合流式细胞术及Western blotting检测PI3K-Akt-mTOR通路对大鼠NPSCs自噬及静息状态的影响
取第三代NPSCs，分别以ML组和SP组为基础，置于37 ℃、5%CO2湿润培养箱中，采用含10%胎牛血清的DMEM/F12完全培养基常规培养，培养至第3天后进行药物干预处理。实验分为6组：ML组、SP组、ML+CQ组、SP+CQ组、ML+MHY1485组、SP+MHY1485组。实验独立重复3次，除ML组和SP组外，ML+CQ组为单层培养的NPSCs加入自噬抑制剂氯喹（CQ）工作液，终浓度10 μmol/L，同条件孵育4 h；SP+CQ组成球培养至第3天的SP组加入CQ工作液，终浓度10 μmol/L，同条件孵育4 h；ML+MHY1485组为单层培养的NPSCs第3天加入mTOR激动剂MHY1485工作液，终浓度20 μmol/L，同条件孵育2 h；SP+MHY1485组成球培养至第3天的NPSCs球状体加入mTOR激动剂MHY1485工作液，终浓度20 μmol/L，同条件孵育2 h。药物孵育结束后，取部分细胞经0.25%胰蛋白酶充分消化为单细胞悬液，参照“1.2.9”方法行流式细胞术检测，分析并统计G0期细胞比例，评价细胞静息状态变化；剩余细胞参照“1.2.13”方法行Western blotting检测，检测指标为p-mTOR、mTOR、LC3B、P62，以GAPDH为内参，计算目的蛋白相对表达量。
1.3　统计学分析
统计学分析采用SPSS 20.0软件完成。所有统计图表采用GraphPad Prism 6.0软件和Origin 9.0软件绘制。所有实验重复3次。数据以x¯±s表示。根据数据的正态分布情况，采用Student’s t检验或单因素方差分析比较组间差异的显著性。P<0.05认为差异具有统计学意义。
2　结果
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2.1　成年人髓核组织成团分布增加及大鼠NPSCs的分离鉴定
对不同年龄段髓核组织HE染色显示：未成年患者髓核细胞呈散在点状分布（图1A），成年患者髓核细胞呈成团簇状分布（图1B），进行多视野观察并统计后提示，成年患者的成团髓核细胞分布模式显著高于未成年患者（P<0.01）。通过组织块培养法从大鼠髓核组织中分离细胞，体外平面培养后呈成纤维细胞样、旋涡状排列（图1C）。流式细胞术检测显示，该细胞高表达间充质干细胞阳性标志物 CD90（99.4%）、CD44（99.8%）、CD73（94.2%），低表达造血干细胞阴性标志物 CD34（0.21%）、CD45（0.45%）、CD11（1.08%），符合间充质干细胞表型（图1D）。三系分化实验证实其干性：成骨诱导后茜素红S染色可见红色钙结节，成软骨诱导后阿利辛蓝染色显示软骨球基质蓝染，成脂诱导后油红O染色可见胞浆内红色脂滴（图1E），表明该细胞具有间充质干细胞的多向分化潜能。
2.2　成团培养促进NPSCs静息并维持干性
确定成团培养时间和大小后，将大鼠NPSCs进行成团培养后，显微观察可见细胞形成稳定的球形细胞团，测得平均直径为（52.3±11.6）μm（图2A）；将该细胞团重新铺板培养，细胞可再次贴壁并恢复生长（图2B），即Re-ML组，提示成团培养的可逆性。采用衰老细胞近红外荧光探针标记后流式检测显示：与ML组相比，SP组的平均荧光强度显著降低，成团培养可减少细胞衰老（图2C）。进一步通过Ki67与PI双染检测细胞周期：与ML组相比，而SP组成团细胞的G0期比例显著升高（图2D）；同时EdU荧光染色结果直观显示：ML组细胞呈现大量红色EdU阳性信号，而SP组成团细胞几乎无EdU阳性信号（图2E）。为进一步确定其干性和静息变化，RT-qPCR结果显示，相较于ML组，SP组NPSCs中静息相关基因P27、SOX2、CD34的mRNA相对表达量均显著升高（P<0.05，图2F）；Western blotting结果与转录水平一致，SP组P27、SOX2的蛋白表达水平明显高于ML组（图2G），提示三维成团培养可有效诱导NPSCs进入静息状态。Western blotting结果显示，SP组OCT4、Nanog的蛋白表达水平显著高于ML组；而Re-ML组经重新贴壁培养后，OCT4、Nanog的蛋白表达水平较SP组明显回落，恢复至接近ML组的水平（图2H）。上述结果表明，成团培养可诱导大鼠NPSCs进入G0期静息状态，静息相关基因与干性相关转录因子的表达也显著上调，而重新贴壁培养可逆转这一效应，成功构建了NPSCs的体外静息模型。
2.3　成团培养抑制NPSCs增殖相关通路并增强自噬相关通路
对ML组和SP组NPSCs进行转录组测序，差异表达基因热图显示2组基因表达模式存在显著分群（图3A）；PCA分析进一步验证了ML与SP组的转录组特征具有明显区分度（图3B）。火山图分析共筛选出显著差异表达基因（|Log2FC|≥1，P<0.05），其中SP组相对于ML组存在大量上调及下调基因（图3C）。GO功能富集分析显示：上调基因主要富集于细胞黏附、自噬调控等生物学过程；下调基因则富集于细胞周期进程、DNA复制等增殖相关条目（图3D）。KEGG通路富集分析表明：SP组上调基因显著富集于自噬通路、ECM-受体相互作用，下调基因则富集于细胞周期、PI3K-Akt信号通路、DNA复制（图3E）。进一步针对核心通路的气泡图分析显示：与ML组相比，SP组中细胞周期、DNA复制相关基因显著下调，PI3K-Akt信号通路相关基因表达受抑，而自噬通路相关基因显著上调（图3F）。PI3K-Akt信号通路和自噬核心通路的差异基因热图结果表明，SP组NPSCs自噬相关基因表达显著上调，PI3K-Akt通路核心基因表达显著下调，2组基因表达谱存在明显差异（图3G）。RT-qPCR验证结果进一步证实，与ML组相比，SP组NPSCs中Pik3cd、Akt1的mRNA表达水平显著降低，Mtor、Atg2a、Atg4c、Atg13的mRNA表达水平显著升高（P<0.05，图3H）。上述结果从转录水平提示，成团培养通过抑制增殖相关通路、激活自噬通路等方式，参与NPSCs静息状态的调控。
2.4　成团培养抑制NPSCs PI3K-Akt-mTOR 信号并增强自噬
Western blotting验证结果显示：SP 组中自噬相关分子LC3-Ⅱ/LC3-Ⅰ比值显著升高（P<0.05），P62表达显著降低（P<0.05，图4A）；同时，PI3K-Akt-mTOR通路中p-Akt、p-mTOR的磷酸化水平显著下调（P<0.05，图4B），总蛋白mTOR、Akt、PI3K的表达也呈降低趋势（P<0.05，图4C）。为进一步验证上述结果，以Ki67为静息状态标志物、LC3B为自噬活性标志物、p-mTOR为 PI3K-Akt-mTOR通路活化标志物，对未成年患者和成年患者髓核组织上述基因进行免疫荧光染色（图4D），未成年患者髓核组织中Ki67、p-mTOR平均荧光强度显著高于成年患者组（P<0.05），而LC3B平均荧光强度显著低于成年患者组（P<0.05）。上述临床样本分子表型变化趋势，与体外NPSCs单层培养组（ML，高Ki67、高p-mTOR、低LC3B）、成球培养组（SP，低Ki67、低p-mTOR、高LC3B）的检测结果完全一致，证实体内髓核细胞成团分布模式与静息状态、自噬激活及PI3K-Akt-mTOR通路抑制的调控关联，与体外实验结果形成相互验证。透射电镜在多个不同视野观察显示：ML组细胞视野内极少观察到自噬溶酶体结构，而SP组细胞内可见大量双层膜包裹的自噬体及自噬溶酶体结构（图4E）。上述结果表明，成团培养通过抑制PI3K-Akt-mTOR通路激活自噬，这是其诱导NPSCs进入静息状态的关键分子机制。
2.5　激活 mTOR 或抑制自噬逆转成团培养诱导的NPSCs静息状态
为明确mTOR通路抑制-自噬激活-细胞静息的因果调控关系，挽救实验结果显示：在ML组和SP组经自噬抑制剂CQ处理后，Western blotting实验显示，SP组P62积累、LC3B-Ⅱ 水平显著升高（P<0.05），明确成团细胞自噬流处于激活状态（图5A）；在SP组中经mTOR激动剂MHY1485处理后，Western blotting实验显示，SP+MHY1485组p-mTOR表达回升、LC3B-Ⅱ/Ⅰ比值降低（P<0.05），自噬被逆转（图5B）；进一步对ML组和SP组细胞周期检测显示，ML+MHY1485组和SP+MHY1485组G0期比例分别较ML组和SP组显著下降（P<0.05，图5C）。上述结果证实：成团培养通过抑制mTOR通路激活自噬以维持干细胞静息状态，而mTOR通路激活可逆转这一表型。
3　讨论
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3.1　临床组织学特征揭示髓核细胞在静息状态的分布规律
IVDD的核心源于NPSCs数量减少、干性耗竭，而成体干细胞的静息和增殖动态平衡是维持干细胞稳态的核心［19-20］。静息状态下的干细胞可通过低代谢、低增殖的方式逃避氧化应激与DNA损伤，从而长期维持干性与再生潜能［21］。本研究通过临床组织学观察发现，成团分布可能是髓核细胞随年龄增长进入静息状态的组织学标志。体内髓核组织的缺氧、低营养微环境可能通过细胞聚集成团形成功能性生态位，维持NPSCs的静息状态以保存其修复潜能。这一临床现象为体外模拟NPSCs静息模型提供了直接的组织学依据，也为理解IVDD中NPSCs功能衰退的机制提供了新视角。
3.2　成团培养构建模拟体内生态位的 NPSCs 功能性静息模型
体外模拟干细胞静息状态的核心是复现体内生态位的调控特征，传统的血清饥饿、接触抑制等方法常伴随干细胞干性耗竭或非特异性损伤［13， 22］。本研究采用的成团培养策略，通过细胞间直接接触、旁分泌信号及局部微环境梯度的影响，使之更接近体内髓核细胞的聚集微环境［23-24］。本实验中成团培养的NPSCs所表现出G0期阻滞、EdU低阳性率和低衰老水平的结果，提示成团培养诱导的静息状态是功能性静息，而非细胞衰老或凋亡的被动结果。此外，成团细胞分散后可快速恢复增殖与分化能力，进一步证实该静息状态的可逆性，这对NPSCs的体外扩增与临床应用具有重要价值。通过成团培养使NPSCs维持静息，既避免了长期贴壁培养导致的干性耗竭，又能在需要时快速激活其修复功能。
3.3　PI3K-Akt-mTOR 通路和自噬通路是成团静息的核心调控通路
从分子机制层面，本研究通过转录组测序与功能验证，明确了PI3K-Akt-mTOR通路抑制和自噬激活的机制，这是成团培养诱导NPSCs静息的核心调控通路。PI3K-Akt-mTOR通路是细胞增殖、代谢与自噬的核心调控节点。在贴壁增殖状态下，该通路的持续激活驱动细胞进入细胞周期，同时通过磷酸化ULK1等分子抑制自噬启动［25-26］，而成团培养可能通过细胞间接触抑制、微环境营养限制等方式，下调了PI3K-Akt-mTOR通路的磷酸化水平，解除了对自噬的负调控，进而通过自噬的激活维持细胞内稳态。本研究所表明自噬相关分子的升高和自噬底物的降解，结合电镜下自噬溶酶体的大量出现，共同证实成团细胞的自噬流处于激活状态。因此，自噬不仅清理了细胞的代谢废物［27］，也帮助调控干细胞的静息代谢。其过程可能是通过降解胞内大分子物质提供基础代谢底物，同时减少活性氧的产生。这与静息NPSCs的低代谢、高存活等特性高度契合［13］。
该模型为IVDD细胞治疗提供了种子细胞功能调控和体内生态位模拟的两种方案，动物体内实验目前可尝试通过经皮穿刺局部注射与Gelma胶结合移植至大鼠退变的椎间盘，若应用于临床，凭借干性保留强、存活时间长、安全风险低等优势，解决传统干细胞移植干性耗竭、存活时间短的瓶颈，同时通过旁分泌调控周围细胞稳态，这为退行性病变的治疗提供思路；但需突破体内成团稳定性（生物交联或支架辅助滞留）、免疫原性控制（如自体细胞来源）、给药方式优化（如匹配穿刺针内径的细胞团大小）等技术挑战，方能推动其从实验研究向临床应用转化。
本研究的发现也存在有待拓展的方向：①缺乏体内实验验证成团静息NPSCs对IVDD的修复效果，后续可通过大鼠尾椎间盘退变模型，评估成团培养的NPSCs移植后的存活、分化及组织修复能力；②未探究细胞间通讯在成团静息中的作用，而体内髓核细胞的成团分布可能依赖细胞间通讯实现同步静息，这一机制的阐明将进一步完善成团培养诱导静息的理论体系。
综上所述，本研究从临床现象出发，构建了基于成团培养的NPSCs静息模型，明确了PI3K-Akt-mTOR通路和自噬通路的核心调控作用，不仅为理解NPSCs的静息机制提供了实验依据，也为IVDD的内源性修复策略提供了新的技术思路。这一技术有望为IVDD的细胞治疗提供更优质的移植源。

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doi: 10.16016/j.2097-0927.202601032
  • 接收时间：2026-01-11
  • 首发时间：2026-10-23
  • 出版时间：2026-05-15
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  • 收稿日期：2026-01-11
  • 修回日期：2026-03-26
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    1陆军军医大学（第三军医大学）第二附属医院骨科，重庆

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黄博，E-mail：
HUANG Bo，E-mail：
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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