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Article(id=1246459856874529480, tenantId=1146029695717560320, journalId=1246415837536497731, issueId=1246459843930903036, articleNumber=null, orderNo=null, doi=10.12307/2025.566, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1722787200000, receivedDateStr=2024-08-05, revisedDate=1734883200000, revisedDateStr=2024-12-23, acceptedDate=1731340800000, acceptedDateStr=2024-11-12, onlineDate=1775108787982, onlineDateStr=2026-04-02, pubDate=1766851200000, pubDateStr=2025-12-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1775108787982, onlineIssueDateStr=2026-04-02, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1775108787982, creator=13701087609, updateTime=1775108787982, updator=13701087609, issue=Issue{id=1246459843930903036, tenantId=1146029695717560320, journalId=1246415837536497731, year='2025', volume='29', issue='36', pageStart='7701', pageEnd='7920', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1775108784853, creator=13701087609, updateTime=1775108852483, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1246460127511991018, tenantId=1146029695717560320, journalId=1246415837536497731, issueId=1246459843930903036, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1246460127511991019, tenantId=1146029695717560320, journalId=1246415837536497731, issueId=1246459843930903036, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=7790, endPage=7796, ext={EN=ArticleExt(id=1246459857201685221, articleId=1246459856874529480, tenantId=1146029695717560320, journalId=1246415837536497731, language=EN, title=Transcription factor NKX2.1 promotes differentiation of induced pluripotent stem cells into lung stem cells, columnId=1246459844752986623, journalTitle=Chinese Journal of Tissue Engineering Research, columnName=Research, runingTitle=null, highlight=null, articleAbstract=
BACKGROUND:

Enhancing the differentiation of induced pluripotent stem cells into lung stem cells is crucial for repairing lung injuries. NKX2.1 is the earliest marker of lung epithelial differentiation and plays a significant regulatory role in lung development. However, the impact of its expression on the differentiation of induced pluripotent stem cells into lung stem cells remains inadequately understood.

OBJECTIVE:

To investigate the effect of NKX2.1 on the differentiation of induced pluripotent stem cells into lung stem cells.

METHODS:

Induced pluripotent stem cells were cultured in vitro. The expression of specific pluripotent stem cell genes was assessed using real-time fluorescence quantitative PCR. NKX2.1 was overexpressed in induced pluripotent stem cells, which were then induced to differentiate into lung stem cells. The expression of FoxA2, SOX9, and P63 was determined via quantitative PCR and immunofluorescence on day 7 of induction of differentiation. The expression of the alveolar marker SPB and SPC was evaluated through immunofluorescence staining on day 7 of induction of differentiation.

RESULTS AND CONCLUSION:

(1) Induced pluripotent stem cells in vitro were tightly packed and showed typical clonoid growth and significantly expressed stem cell-specific genes OCT-4, SOX2, and NANOG. (2) Compared with the non-transfected control group, the expression of NKX2.1 in human induced pluripotent stem cells was significantly increased in the NKX2.1 overexpression group (P < 0.000 1). (3) Seven days after induction of differentiation, compared with the non-transfected control group, the expression of lung stem cell-related markers FoxA2, SOX9, and P63 was significantly increased in the NKX2.1 overexpression group (P < 0.000 1). (4) Thirteen days after induction of differentiation, compared with the non-transfected control group, the fluorescence intensity of alveolar cell marker molecules SPB and SPC increased significantly in the overexpression NKX2.1 group. The results show that NKX2.1 can promote the differentiation of induced pluripotent stem cells into lung stem cells.

, correspAuthors=null, authorNote=null, correspAuthorsNote=
Dong Mingqing, MD, Professor, Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China
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背景：

如何促进诱导多能干细胞向肺干细胞分化对肺损伤修复具有重要意义。NKX2.1是肺上皮分化过程中最早的标志物，在肺发育过程中发挥重要调控作用，然而其表达对诱导多能干细胞向肺干细胞分化的影响还知之甚少。

目的：

探究NKX2.1对诱导多能干细胞向肺干细胞方向分化的影响。

方法：

体外培养人诱导多能干细胞，采用实时荧光定量PCR和免疫荧光检测多能干细胞特异基因的表达；通过瞬时转染向人诱导多能干细胞中过表达NKX2.1，再诱导其向肺干细胞方向分化，诱导分化7 d采用实时荧光定量PCR、免疫荧光检测FoxA2、SOX9和P63的表达，诱导分化13 d采用免疫荧光检测肺泡细胞标记分子SPB和SPC的表达。

结果与结论：

①诱导多能干细胞紧密集结呈典型克隆样生长，显著高表达干细胞特异表达基因OCT-4、SOX2和NANOG；②与未转染对照组相比，过表达NKX2.1组人诱导多能干细胞中NKX2.1表达显著升高（P < 0.000 1）；③诱导分化7 d，与未转染对照组相比，过表达NKX2.1组肺干细胞相关标记物FoxA2、SOX9和P63的表达显著升高（P < 0.000 1）；④诱导分化13 d，与未转染对照组相比，过表达NKX2.1组肺泡细胞标记分子SPB和SPC荧光强度显著增加。结果表明NKX2.1可以促进诱导多能干细胞向肺干细胞方向分化。

, correspAuthors=null, authorNote=null, correspAuthorsNote=
董明清，博士，教授，成都市第五人民医院医学研究与转化中心，四川省成都市　611130
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作者贡献：

实验设计董明清，实验实施为邓丽、刘洋、王慧，实验评估为董明清、杨秋，数据统计为邓丽、刘洋。

Deng Li, PhD, Assistant researcher, Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China

邓丽，女，1988年生，云南省昭通市人，汉族，2023年电子科技大学毕业，博士，助理研究员，主要从事干细胞定向分化和组织工程相关基础研究。

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Deng Li, PhD, Assistant researcher, Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China

邓丽，女，1988年生，云南省昭通市人，汉族，2023年电子科技大学毕业，博士，助理研究员，主要从事干细胞定向分化和组织工程相关基础研究。

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Deng Li, PhD, Assistant researcher, Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China

邓丽，女，1988年生，云南省昭通市人，汉族，2023年电子科技大学毕业，博士，助理研究员，主要从事干细胞定向分化和组织工程相关基础研究。

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图注：图中A为人诱导多能干细胞形态（标尺为100 μm）；B为实时荧光定量PCR检测干细胞多能性因子NANOG、SOX2和OCT4 mRNA表达，与成纤维细胞相比，aP < 0.000 1；C为免疫荧光检测干细胞多能性因子NANOG、SOX2和OCT4的表达（标尺为100 μm）。

, figureFileSmall=amjRiLdTx+PkY4JvStoeSA==, figureFileBig=TSXBCF3faGWHAASxDcCpJA==, tableContent=null), ArticleFig(id=1246459873588830404, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=EN, label=Figure 3, caption=Effect of overexpression of NKX2.1 plasmid in human induced pluripotent stem cells, figureFileSmall=55bhbiSPQR8TlPhlepKT6A==, figureFileBig=FmMnXz1OjeXux/iSvihnXA==, tableContent=null), ArticleFig(id=1246459873689493704, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=CN, label=图3, caption=人诱导多能干细胞过表达NKX2.1质粒的效果

图注：图中A，B为转染NKX2.1过表达质粒48，96 h后人诱导多能干细胞中NKX2.1 mRNA表达；C为转染NKX2.1过表达质粒72 h后人诱导多能干细胞中NKX2.1蛋白表达。与未转染对照组相比，aP < 0.000 1。

, figureFileSmall=55bhbiSPQR8TlPhlepKT6A==, figureFileBig=FmMnXz1OjeXux/iSvihnXA==, tableContent=null), ArticleFig(id=1246459873794351310, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=EN, label=Figure 4, caption=Effect of overexpression of NKX2.1 on the expression of FoxA2, SOX9, and P63 during differentiation of human induced pluripotent stem cells into lung stem cells, figureFileSmall=ojJgFHCNqZ5Bh9zwoj6Ylw==, figureFileBig=3TTfB0rWEZbnCefMiLjfAQ==, tableContent=null), ArticleFig(id=1246459873907597527, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=CN, label=图4, caption=过表达NKX2.1对人诱导多能干细胞向肺干细胞分化过程中FoxA2、SOX9、P63表达的影响

图注：图中A为RT-qPCR检测FoxA2、SOX9和P63 mRNA表达；B，C为免疫荧光检测FoxA2、SOX9和P63蛋白表达（标尺为100 μm）；D为荧光强度半定量统计结果。hiPSCs为未诱导的人诱导多能干细胞，NKX2.1(+）为诱导分化7 d的过表达NKX2.1人诱导多能干细胞，NKX2.1(-）为诱导分化7 d的未转染人诱导多能干细胞。aP < 0.01。

, figureFileSmall=ojJgFHCNqZ5Bh9zwoj6Ylw==, figureFileBig=3TTfB0rWEZbnCefMiLjfAQ==, tableContent=null), ArticleFig(id=1246459874020843739, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=EN, label=Figure 5, caption=Immunofluorescence staining of SPB and SPC after 13 days of differentiation of human induced pluripotent stem cells overexpressing NKX2.1 into lung stem cells, figureFileSmall=f+GidmNJa0uAaccS0p1alg==, figureFileBig=mzbE1O9j96WLkA0RGSL0Cw==, tableContent=null), ArticleFig(id=1246459874146672866, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=CN, label=图5, caption=过表达NKX2.1人诱导多能干细胞向肺干细胞分化13 d后SPB、SPC免疫荧光染色

图注：图中A-F为SPB、SPC免疫荧光染色（标尺为100 μm），其中A，D为仅二抗孵育的抗体对照组；B，E为诱导分化13 d的未转染人诱导多能干细胞；C，F为诱导分化13 d的过表达NKX2.1人诱导多能干细胞；G，H为SPB和SPC荧光强度半定量统计结果。与未转染对照组相比，aP < 0.001。

, figureFileSmall=f+GidmNJa0uAaccS0p1alg==, figureFileBig=mzbE1O9j96WLkA0RGSL0Cw==, tableContent=null), ArticleFig(id=1246459874268307689, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=EN, label=Table 1, caption=

RT-qPCR primer sequences

, figureFileSmall=null, figureFileBig=null, tableContent=
基因引物序列（5'-3'）
GAPDHForward：ACA TCG CTC AGA CAC CAT G
 Reverse：TGT AGT TGA GGT CAA TGA AGG G
OCT4Forward：TCT CCC ATG CAT TCA AAC TGA G
 Reverse：CCT TTG TGT TCC CAA TTC CTT C
NANOGForward：GAA ATA CCT CAG CCT CCA GC
 Reverse：GCG TCA CAC CAT TGC TAT TC
SOX2Forward：TAC AGC ATG TCC TAC TCG CAG
 Reverse：GAG GAA GAG GTA ACC ACA AGG G
NKX2.1Forward：CTG CTC CAC CTT GCT ATA CG
 Reverse：GTT AAG AAA AGT CGA AGC GCG
FoxA2Forward：TTT TAA ACT GCC ATG CAC TCG
 Reverse：TTC ATG TTG CTC ACG GAG G
SOX9Forward：ACT TGC ACA ACG CCG AG
 Reverse：CTG GTA CTT GTA ATC CGG GTG
P63Forward：TTC GGA CAG TAC AAA GAA CGG
 Reverse：GCA TTT CAT AAG TCT CAC GGC
), ArticleFig(id=1246459874377359596, tenantId=1146029695717560320, journalId=1246415837536497731, articleId=1246459856874529480, language=CN, label=表1, caption=

RT-qPCR引物

, figureFileSmall=null, figureFileBig=null, tableContent=
基因引物序列（5'-3'）
GAPDHForward：ACA TCG CTC AGA CAC CAT G
 Reverse：TGT AGT TGA GGT CAA TGA AGG G
OCT4Forward：TCT CCC ATG CAT TCA AAC TGA G
 Reverse：CCT TTG TGT TCC CAA TTC CTT C
NANOGForward：GAA ATA CCT CAG CCT CCA GC
 Reverse：GCG TCA CAC CAT TGC TAT TC
SOX2Forward：TAC AGC ATG TCC TAC TCG CAG
 Reverse：GAG GAA GAG GTA ACC ACA AGG G
NKX2.1Forward：CTG CTC CAC CTT GCT ATA CG
 Reverse：GTT AAG AAA AGT CGA AGC GCG
FoxA2Forward：TTT TAA ACT GCC ATG CAC TCG
 Reverse：TTC ATG TTG CTC ACG GAG G
SOX9Forward：ACT TGC ACA ACG CCG AG
 Reverse：CTG GTA CTT GTA ATC CGG GTG
P63Forward：TTC GGA CAG TAC AAA GAA CGG
 Reverse：GCA TTT CAT AAG TCT CAC GGC
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转录因子NKX2.1促进诱导多能干细胞向肺干细胞的分化
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邓丽 , 刘洋 , 王慧 , 杨秋 , 董明清
中国组织工程研究 | 研究原著 2025,29(36): 7790-7796
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中国组织工程研究 | 研究原著 2025, 29(36): 7790-7796
转录因子NKX2.1促进诱导多能干细胞向肺干细胞的分化
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邓丽, 刘洋, 王慧, 杨秋, 董明清
作者信息
  • 成都市第五人民医院医学研究与转化中心，四川省成都市　611130

    • Deng Li, PhD, Assistant researcher, Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China

    邓丽，女，1988年生，云南省昭通市人，汉族，2023年电子科技大学毕业，博士，助理研究员，主要从事干细胞定向分化和组织工程相关基础研究。

通讯作者:

董明清，博士，教授，成都市第五人民医院医学研究与转化中心，四川省成都市　611130
Transcription factor NKX2.1 promotes differentiation of induced pluripotent stem cells into lung stem cells
Li Deng, Yang Liu, Hui Wang, Qiu Yang, Mingqing Dong
Affiliations
  • Center for Medicine Research and Translation, Chengdu Fifth People's Hospital, Chengdu 611130, Sichuan Province, China
出版时间: 2025-12-28 doi: 10.12307/2025.566
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摘要
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背景：

如何促进诱导多能干细胞向肺干细胞分化对肺损伤修复具有重要意义。NKX2.1是肺上皮分化过程中最早的标志物，在肺发育过程中发挥重要调控作用，然而其表达对诱导多能干细胞向肺干细胞分化的影响还知之甚少。

目的：

探究NKX2.1对诱导多能干细胞向肺干细胞方向分化的影响。

方法：

体外培养人诱导多能干细胞，采用实时荧光定量PCR和免疫荧光检测多能干细胞特异基因的表达；通过瞬时转染向人诱导多能干细胞中过表达NKX2.1，再诱导其向肺干细胞方向分化，诱导分化7 d采用实时荧光定量PCR、免疫荧光检测FoxA2、SOX9和P63的表达，诱导分化13 d采用免疫荧光检测肺泡细胞标记分子SPB和SPC的表达。

结果与结论：

①诱导多能干细胞紧密集结呈典型克隆样生长，显著高表达干细胞特异表达基因OCT-4、SOX2和NANOG；②与未转染对照组相比，过表达NKX2.1组人诱导多能干细胞中NKX2.1表达显著升高（P < 0.000 1）；③诱导分化7 d，与未转染对照组相比，过表达NKX2.1组肺干细胞相关标记物FoxA2、SOX9和P63的表达显著升高（P < 0.000 1）；④诱导分化13 d，与未转染对照组相比，过表达NKX2.1组肺泡细胞标记分子SPB和SPC荧光强度显著增加。结果表明NKX2.1可以促进诱导多能干细胞向肺干细胞方向分化。

诱导多能干细胞  /  肺干细胞  /  NKX2.1  /  定型内胚层  /  前肠内胚层  /  肺损伤  /  工程化干细胞
BACKGROUND:

Enhancing the differentiation of induced pluripotent stem cells into lung stem cells is crucial for repairing lung injuries. NKX2.1 is the earliest marker of lung epithelial differentiation and plays a significant regulatory role in lung development. However, the impact of its expression on the differentiation of induced pluripotent stem cells into lung stem cells remains inadequately understood.

OBJECTIVE:

To investigate the effect of NKX2.1 on the differentiation of induced pluripotent stem cells into lung stem cells.

METHODS:

Induced pluripotent stem cells were cultured in vitro. The expression of specific pluripotent stem cell genes was assessed using real-time fluorescence quantitative PCR. NKX2.1 was overexpressed in induced pluripotent stem cells, which were then induced to differentiate into lung stem cells. The expression of FoxA2, SOX9, and P63 was determined via quantitative PCR and immunofluorescence on day 7 of induction of differentiation. The expression of the alveolar marker SPB and SPC was evaluated through immunofluorescence staining on day 7 of induction of differentiation.

RESULTS AND CONCLUSION:

(1) Induced pluripotent stem cells in vitro were tightly packed and showed typical clonoid growth and significantly expressed stem cell-specific genes OCT-4, SOX2, and NANOG. (2) Compared with the non-transfected control group, the expression of NKX2.1 in human induced pluripotent stem cells was significantly increased in the NKX2.1 overexpression group (P < 0.000 1). (3) Seven days after induction of differentiation, compared with the non-transfected control group, the expression of lung stem cell-related markers FoxA2, SOX9, and P63 was significantly increased in the NKX2.1 overexpression group (P < 0.000 1). (4) Thirteen days after induction of differentiation, compared with the non-transfected control group, the fluorescence intensity of alveolar cell marker molecules SPB and SPC increased significantly in the overexpression NKX2.1 group. The results show that NKX2.1 can promote the differentiation of induced pluripotent stem cells into lung stem cells.

induced pluripotent stem cell  /  lung stem cell  /  NKX2.1  /  definitive endoderm  /  foregut endoderm  /  lung injury  /  engineered stem cell
邓丽, 刘洋, 王慧, 杨秋, 董明清. 转录因子NKX2.1促进诱导多能干细胞向肺干细胞的分化. 中国组织工程研究, 2025 , 29 (36) : 7790 -7796 . DOI: 10.12307/2025.566
Li Deng, Yang Liu, Hui Wang, Qiu Yang, Mingqing Dong. Transcription factor NKX2.1 promotes differentiation of induced pluripotent stem cells into lung stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025 , 29 (36) : 7790 -7796 . DOI: 10.12307/2025.566
正文
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0　引言　Introduction
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肺损伤及肺纤维化等难治性肺病的发病机制复杂，目前临床药物特异性和靶向性较差，治疗效果并不理想[1-3]，因此迫切需要开发治疗这类疾病的新药物和治疗方法。成体干细胞和祖细胞在维持特定器官的细胞稳态中发挥着重要作用，同时也参与疾病状态下的组织修复[4-6]。通过将器官特异性干细胞引入体内，用于修复、补充或替代受损细胞的方法，即干细胞治疗，为传统治疗效果不佳的难治性疾病提供了新希望。
在肺部损伤修复过程中，具有肺上皮分化潜能的肺干细胞进入肺组织后能够分化为肺部结构细胞（如气道上皮细胞和肺泡上皮细胞），从而替代、补充和修复受损的肺细胞，在肺损伤治疗中表现出了良好的应用前景[7-8]。然而，成体肺脏中肺干细胞数量较少、取材过程具有创伤性、在慢性肺病状态下其功能可能会受到影响等因素限制了肺干细胞治疗的深入研究和临床应用[1]。诱导多能干细胞（induced pluripotent stem cells，iPSCs）的出现为获取包括肺干细胞在内的器官特异性干细胞提供了新途径。诱导多能干细胞可以通过重编程患者的体细胞获得，具有与胚胎干细胞相似的多能性和无限增殖能力。通过模拟器官发育过程中的信号作用模式，可以将诱导多能干细胞诱导分化为不同发育阶段的器官特异性干细胞，实现个性化的干细胞靶向治疗[9-11]。目前，研究人员已能通过体外一系列步骤再现胚胎发育的关键阶段，即定向分化，实现胚胎干细胞、间充质干细胞和诱导多能干细胞等向感兴趣的细胞和组织谱系方向分化[12-16]。ALBER等[17]构建了一种Tbx4肺增强子示踪的小鼠诱导多能干细胞，定向诱导其进入早期肺间质，这些诱导多能干细胞衍生的肺间充质祖细胞与原代小鼠胚胎肺间充质具有相同的转录特征，并且能够分化为更成熟的间充质谱系。KANAGAKI等[18]通过Wnt信号抑制诱导多能干细胞向肺泡Ⅰ型细胞定向分化。然而，这些方法都存在诱导周期长且获得肺干细胞数量有限等限制，未能满足临床应用需求。因此，探究肺干细胞分化的机制，寻找更快、更利于肺干细胞分化的方法具有重要现实意义。
NKX2.1，也被称为TTF-1，是由位于人类第14号染色体上的基因编码的一种转录因子，参与调节形态发生，特别是甲状腺、肺和大脑发育过程中基因的表达[17]。研究表明，NKX2.1是肺上皮分化过程中最早的标志物，它的缺失会导致肺发育不全[18-21]。急性损伤后肺上皮细胞的再生需要多种细胞的协调来形成形态复杂的肺泡气体交换表面，而NKX2.1是肺泡上皮祖细胞的关键调控因子。NKX2.1表观遗传的动态维持是控制肺泡上皮祖细胞中选择性祖细胞活性的关键因素。在成年肺再生过程中，Wnt响应的肺泡上皮祖细胞增殖并转化为肺泡Ⅰ型/Ⅱ型细胞的过程受NKX2.1差异性活性的调控[22-27]。然而，通过差异表达NKX2.1以调节人诱导多能干细胞向肺干细胞方向分化的研究尚不明确。因此，该研究尝试在人诱导多能干细胞中过表达NKX2.1，旨在探究NKX2.1表达对人诱导多能干细胞向肺干细胞方向分化的影响。
1　材料和方法　Materials and methods
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1.1　设计
诱导多能干细胞向肺干细胞方向分化实验。
1.2　时间及地点
实验于2023年1月至2024年6月在成都市第五人民医院医学研究与转化中心完成。
1.3　材料
1.3.1　细胞株
成纤维细胞、人诱导多能干细胞株购自四川康克俞生物科技有限公司。
1.3.2　实验试剂和仪器
人诱导多能干细胞维持自我更新完全培养基、Vitronectin包被蛋白、细胞冻存液、细胞消化液（中国中盛溯源生物科技有限公司）；DPBS、DMEM/F12、GlutaMax-supplemented DMEM/F12、RPMI 1640（美国GIBICO公司）；转染试剂Lipofectamine 2000（美国ThermoFisher公司）；Activin A（美国R&D Systems公司）；Noggin、成纤维生长因子7、成纤维生长因子4和骨形态发生蛋白4（美国Stemimmune LLC公司）；SB431542（英国Tocris公司）；CHIR99021（加拿大Stem Cell Technologies公司）；SPB抗体、FoxA2抗体、SOX9抗体、P63抗体、SPC抗体、NKX2.1抗体（中国博奥森生物公司）；Anti-Mouse/Rabbit荧光二抗、GAPDH抗体、DAPI（中国华安生物公司）；总RNA提取试剂盒、RNA反转录试剂盒、实时荧光定量PCR试剂盒（中国诺维赞生物公司）；实时荧光定量PCR引物（中国擎科生物公司）；NKX2.1过表达质粒（中国生工公司）；倒置相差荧光显微镜（日本奥林巴斯）。
1.4　实验方法
1.4.1　人诱导多能干细胞培养
提前加入Vitronectin包被蛋白于6孔板中孵育过夜，传代前弃包被蛋白。将人诱导多能干细胞用DPBS轻洗1次，加入细胞消化液至细胞变圆时终止消化，3 500 r/min离心5 min，弃上清，细胞沉淀用人诱导多能干细胞培养基重悬，以3×105/cm2密度接种于处理过的6孔板中，加入人诱导多能干细胞培养基，置于37 ℃、体积分数5% CO2培养箱中，每天换液1次，每两三天传代1次，通过光学显微镜观察细胞及克隆形态，RT-qPCR检测OCT4、NANOG和SOX2 mRNA表达水平，引物序列见表1；免疫荧光观察OCT4、NANOG和SOX2蛋白表达水平。
1.4.2　瞬时转染建立过表达NKX2.1人诱导多能干细胞
人诱导多能干细胞分为2组：未转染对照组，过表达NKX2.1组。根据说明书，采用Lipofectamine 2000将NKX2.1过表达质粒转染入第15代人诱导多能干细胞中，转染4-6 h后更换新鲜完全培养基，培养48，96 h采用RT-qPCR检测NKX2.1 mRNA表达，引物序列见表1；培养72 h采用Western blot检测NKX2.1蛋白表达。
1.4.3　人诱导多能干细胞向肺干细胞方向分化
参照GHAEDI等[28]和YAMAMOTO等[29]的方法，采用单层细胞贴壁法对未转染对照组和过表达NKX2.1组人诱导多能干细胞进行诱导分化。人诱导多能干细胞消化后以1.5×105/cm2密度接种于Vitronectin包被蛋白处理过的6孔板中，24 h后将细胞培养基更换为RPMI 1640添加Activin A（100 ng/mL）培养3 d，更换培养基为GlutaMax-supplemented DMEM/F12添加Noggin（200 ng/mL）和SB431542（10 μmol/L）培养4 d，更换培养基为GlutaMax-supplemented DMEM/F12添加CHIR99021（3 μmol/L）、成纤维生长因子7（10 ng/mL）、成纤维生长因子4（10 ng/mL）和骨形态发生蛋白4（20 ng/mL）培养6 d。诱导方案如图1所示。
1.4.4　Western blot检测
人诱导多能干细胞转染后培养72 h检测NKX2.1蛋白表达，细胞刮于冰上收集细胞，3 500 r/min离心弃上清，加适量细胞裂解缓冲液重悬置于冰上裂解25 min，每隔5 min斡旋1次，4 ℃、12 000×g离心10 min，SDS上样缓冲液重悬制备样本，通过10%的SDS-PAGE电泳分离蛋白后，转移到硝酸纤维膜上，5% BSA室温封闭45 min，加入5% BSA稀释的一抗（GAPDH 1∶2 000，NKX2.1 1∶1 000），4 ℃孵育过夜，室温摇床TBST洗膜5次，5 min/次，加入2.5% BSA（1∶5 000）稀释HRP酶标抗兔/小鼠二抗抗体室温孵育1 h，采用ECL试剂盒对膜上目标蛋白进行显影。
1.4.5　实时荧光定量PCR（RT-qPCR）检测
未转染人诱导多能干细胞用于检测OCT4、NANOG和SOX2 mRNA表达；人诱导多能干细胞转染后培养48，96 h检测NKX2.1 mRNA表达；人诱导多能干细胞分化培养7 d检测FoxA2、SOX9和P63 mRNA表达。操作步骤如下：弃培养基，根据总RNA提取试剂盒使用说明书提取细胞RNA并测定浓度，再利用反转录试剂盒得到样本cDNA，按照RT-qPCR说明书操作，以人GAPDH为内参，通过ΔΔCt法对各基因表达进行相对定量分析。引物序列见表1。
1.4.6　细胞免疫荧光
未转染人诱导多能干细胞进行OCT4、NANOG和SOX2免疫荧光染色；人诱导多能干细胞分化培养7 d进行FoxA2、SOX9和P63免疫荧光染色；人诱导多能干细胞分化培养13 d进行SPB、SPC免疫荧光染色。操作步骤如下：弃培养基，在室温下用PBS轻洗两三次（5 min/次），加入40 g/L多聚甲醛室温固定30 min，PBS洗两三次（5 min/次），0.25%免疫染色通透液TritonX-100室温孵育30 min，PBS轻洗两三次（5 min/次），封闭液3%BSA室温孵育1 h，加入一抗抗体（由1% BSA配置，SPB、SPC、FoxA2、SOX9、P63按照1∶300稀释，OCT4、NANOG和SOX2按照1∶500稀释）4 ℃孵育过夜，弃一抗，PBS轻洗两三次（5 min/次），加入荧光二抗（由1% BSA按照1∶1 000稀释）室温避光孵育1 h，DAPI染色工作液避光室温染色10 min，PBS洗两三次（5 min/次），荧光显微镜下观察并拍照。通过Image J软件对荧光强度进行半定量统计分析。
1.5　主要观察指标
①人诱导多能干细胞表型及干性标记分子的表达；②人诱导多能干细胞向肺干细胞分化过程中相关标记分子的表达。
1.6　统计学分析
实验数据利用GraphPad Prism 8.4.0分析，数据以表示，进行t检验或单因素方差分析，P < 0.05为差异有显著性意义，文章统计学方法已经通过成都市第五人民医院专家审核。
2　结果　Results
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2.1　人诱导多能干细胞表型及干性标记分子的表达
如图2A所示，人诱导多能干细胞体外传代培养后，显微镜下观察细胞以紧密集结形成典型“干细胞簇”的表型，表明细胞状态良好。如图2B所示，人诱导多能干细胞中干性标记分子OCT4、NANOG和SOX2的mRNA表达水平较成纤维细胞显著增加（P < 0.000 1），表明人诱导多能干细胞干性维持良好。如图2C所示，免疫荧光检测人诱导多能干细胞中显著表达NANOG、SOX2和OCT4蛋白。
2.2　瞬时转染获得过表达NKX2.1的人诱导多能干细胞
通过Lipofectamine 2000转染试剂将NKX2.1过表达质粒瞬时转染入人诱导多能干细胞后，分别于48，96 h采用RT-qPCR检测NKX2.1 mRNA表达，72 h采用Western blot检测NKX2.1的蛋白表达，见图3。与未转染对照组相比，过表达NKX2.1组细胞中NKX2.1 mRNA和蛋白表达明显升高，差异有显著性意义（P < 0.000 1），提示成功转染NKX2.1过表达质粒。
2.3　NKX2.1过表达对人诱导多能干细胞向肺干细胞分化的影响
人诱导多能干细胞向肺干细胞方向分化要经历定形内胚层、腹部前肠内胚层等阶段[29]。与正常的人诱导多能干细胞相比，诱导7 d后的未转染对照组和过表达NKX2.1组人诱导多能干细胞中均显著高表达肺干细胞相关标记物FoxA2、SOX9、P63（P < 0.000 1）；与未转染对照组相比，过表达NKX2.1组FoxA2、SOX9、P63的mRNA表达明显升高（P < 0.000 1），见图4A。此外，免疫荧光染色显示，诱导7 d时过表达NKX2.1组细胞中显著表达FoxA2、SOX9和P63蛋白，见图4B-D。
2.4　NKX2.1过表达可促进人诱导多能干细胞向肺泡分化
为进一步证实NKX2.1过表达对人诱导多能干细胞向肺泡方向分化的影响，诱导分化13 d通过细胞免疫荧光检测肺泡细胞标记分子SPB和SPC的表达。如图5所示，与未转染对照组相比，NKX2.1过表达组SPB和SPC荧光强度明显升高，提示NKX2.1过表达可以显著促进人诱导多能干细胞向肺泡方向分化。
3　讨论　Discussion
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虽然肺移植是终末期肺病患者的一种可能治疗方法，但它受到供肺供应不足、手术创伤和免疫并发症等限制[30]。每年有许多患者在供肺等待名单上或因移植并发症而死亡[31-33]。体外诱导或分离的成人肺干/祖细胞移植成为替代全器官移植有前景的方法[34-36]。然而受体外培养周期长、扩增足够数量细胞困难等限制，寻找更快、更利于肺干细胞分化的方法尤为重要。目前常用的人诱导多能干细胞向肺干细胞分化的诱导方案多为16-21 d不等，根据肺干细胞发生的分子机制和特点将该过程简单的分为定形内胚层、腹部前肠内胚层和肺前体多能细胞3个阶段[37]。FoxA2、SOX9和P63为分化前期阶段所表达的特异性标记蛋白，而SPB则是分化后期阶段Ⅱ型肺泡多能细胞所表达的一种特异性生物标记物[38]。在肺的发育过程中，肺上皮和间充质前体相互作用，启动了复杂的器官发生发育程序，内胚层谱系中表达的转录因子FOXA2常用以标记分化的定形内胚层阶段[39-40]。SOX9是srybox-containing（SOX）蛋白家族的一员，P63为p53转录因子家族的成员之一，参与肺干细胞分化的调控，是早期肺祖细胞标志物[41]。
在人诱导多能干细胞衍生的肺祖细胞体外分化过程中，多个信号通路参与其中，它们在细胞分化的不同阶段被激活或抑制，最终实现分化[42]。KONISHI等[43]发现抑制Wnt激活可促进原始NKX2-1+祖细胞中近端上皮细胞的快速出现，而维持Wnt信号可促进远端上皮细胞的形成，早期抑制转化生长因子β信号途径是肺干细胞分化所必须的，作为肺上皮分化过程中最早标志物的转录因子NKX2.1可与Wnt、转化生长因子β、Notch等多种信号通络相互作用，调控肺干细胞分化下游相关标志物的表达。为了探究过表达NKX2.1对肺干细胞分化的影响，在过表达NKX2.1的条件下，通过在不同时间段添加转化生长因子β受体激酶抑制剂（SB431542）或Wnt/β-catenin信号通路激活剂（CHIR99021）等将人诱导多能干细胞向肺干细胞方向分化，分别在7 d和13 d时检测了不同阶段特异性标记蛋白的表达。诱导分化7 d，过表达NKX2.1人诱导多能干细胞中肺干细胞早期标志物FoxA2、SOX9和P63的表达显著高于未转染的人诱导多能干细胞。以往研究中，FoxA2、P63等早期肺干细胞标志物通常在诱导分化10-14 d高表达[28]，表明过表达NKX2.1可以显著提前启动人诱导多能干细胞向肺干细胞分化。同时，诱导分化13 d，过表达NKX2.1人诱导多能干细胞中明显高表达晚期肺泡细胞标记分子SPB和SPC，表明细胞分化明显提前。以上结果提示，人诱导多能干细胞中NKX2.1过表达与其向肺干细胞方向分化呈正相关。
综上，该研究结果表明过表达NKX2.1可以缩短人诱导多能干细胞向肺干细胞方向分化的时间，并显著提高分化过程中标记分子的表达，细胞免疫荧光结果同样证明过表达NKX2.1可以更高效地促进人诱导多能干细胞向肺泡细胞分化。但该实验也具有一定的局限性：首先，肺干细胞可分化为不同类型肺泡细胞和支持细胞等，增加诱导后细胞向终末分化肺相关细胞分化能力的检测可以更加全面评价过表达NKX2.1对人诱导干细胞向肺干细胞分化的影响；其次，肺干细胞是肺损伤修复最有前景的细胞来源，构建肺损伤动物模型并用过表达NKX2.1人诱导多能干细胞诱导的肺干细胞进行治疗，从动物水平评价过表达NKX2.1人诱导多能干细胞诱导的肺干细胞功能；最后，NKX2.1是如何调控人诱导干细胞向肺干细胞方向分化仍有待进一步研究。
基金
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  • 四川省科技厅自然科学基金项目(2023NSFSC0531)
  • 成都市高水平临床重点专科建设项目(KYJJ2021-27)
  • 成都中医药大学“杏林学者”医院专项(XJ2023010001)
文献
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2025年第29卷第36期
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doi: 10.12307/2025.566
  • 接收时间：2024-08-05
  • 首发时间：2026-04-02
  • 出版时间：2025-12-28
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  • 收稿日期：2024-08-05
  • 修回日期：2024-12-23
  • 录用日期：2024-11-12
基金
Natural Science Foundation of Sichuan Provincial Department of Science and Technology(2023NSFSC0531)
四川省科技厅自然科学基金项目(2023NSFSC0531)
Chengdu High-Level Clinical Key Specialty Construction Project(KYJJ2021-27)
成都市高水平临床重点专科建设项目(KYJJ2021-27)
Chengdu University of Traditional Chinese Medicine “Xinglin Scholar” Hospital Special Project(XJ2023010001)
成都中医药大学“杏林学者”医院专项(XJ2023010001)
作者信息
    成都市第五人民医院医学研究与转化中心，四川省成都市　611130

通讯作者:

董明清，博士，教授，成都市第五人民医院医学研究与转化中心，四川省成都市　611130
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https://castjournals.cast.org.cn/joweb/zgzzgcyj/CN/10.12307/2025.566
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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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