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北京大学学报（医学版） ›› 2025, Vol. 57 ›› Issue (2): 227-236. doi: 10.19723/j.issn.1671-167X.2025.02.002

• 论著 • 上一篇    下一篇

siRNA沉默NLK基因促进神经化组织工程骨再生

李梦迪1, 雷蕾2, 刘中宁1,*(), 李健1, 姜婷1,*()   

  1. 1. 北京大学口腔医学院·口腔医院修复科，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，北京 100081
    2. 首都医科大学附属北京友谊医院口腔科，北京 100050
  • 收稿日期:2021-10-10 出版日期:2025-04-18 发布日期:2025-04-12
  • 通讯作者: 刘中宁,姜婷 E-mail:lzn45@163.com;jt_ketizu@163.com
  • 基金资助:
    国家自然科学基金(81771045);国家自然科学基金(82170928);北京市科技新星计划项目(Z191100001119096)

Gene silencing of Nemo-like kinase promotes neuralized tissue engineered bone regeneration

Mengdi LI1, Lei LEI2, Zhongning LIU1,*(), Jian LI1, Ting JIANG1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
    2. Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
  • Received:2021-10-10 Online:2025-04-18 Published:2025-04-12
  • Contact: Zhongning LIU, Ting JIANG E-mail:lzn45@163.com;jt_ketizu@163.com
  • Supported by:
    the National Natural Science Foundation of China(81771045);the National Natural Science Foundation of China(82170928);the Beijing Nova Program of Science and Technology(Z191100001119096)

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摘要：

目的: 探讨沉默及过表达Nemo样激酶(Nemo-like kinase，NLK)基因对人骨髓间充质干细胞(human mesenchymal stem cells，hBMSCs)神经向分化的作用，并探究小干扰RNA(small interfering RNA，siRNA)沉默NLK基因表达对促进神经化组织工程骨再生的影响。方法: 在hBMSCs中分别转染siRNA沉默NLK基因(实验组转染沉默NLK基因的siRNA，对照组转染对照siRNA并记为阴性对照组)或慢病毒过表达NLK基因(实验组感染过表达NLK基因的慢病毒，对照组感染空载体慢病毒并记为空载体组)，之后通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qPCR)、蛋白质印迹实验(Western blot)、免疫荧光实验检测敲低、过表达NLK基因对hBMSCs神经向分化的影响。采用6周龄雄性BALB/C nu/nu裸鼠构建皮下异位成骨模型，分为以下4组：① β-磷酸三钙(β-tricalcium phosphate, β-TCP)组，② β-TCP+ hBMSCs成骨诱导组，③ β-TCP+siRNA-阴性对照(negative control, NC) 转染的hBMSCs成骨诱导组，④ β-TCP+ siRNA-NLK转染的hBMSCs成骨诱导组。术后4周获取标本，通过组织形态学染色检测裸鼠体内成骨成神经情况，使用独立样本t检验进行统计学分析。结果: siRNA沉默hBMSCs的NLK基因后，神经相关基因βⅢ微管蛋白(class Ⅲ β-tubulin，TUBB3)、微管相关蛋白2(microtubule association protein-2，MAP2)、可溶性蛋白-100(soluble protein-100，S100)、巢蛋白(nestin，NES)、NG2蛋白聚糖(NG2 proteoglycan，NG2)及降钙素基因相关肽(calcitonin gene-related peptide, CGRP)表达较阴性对照组显著升高(P < 0.05)，TUBB3、MAP2等蛋白表达也升高。慢病毒过表达NLK基因后，与空载体组相比，神经相关基因TUBB3、MAP2、S100及NG2表达水平均显著下降(P < 0.05)，TUBB3蛋白表达降低。构建裸鼠皮下异位成骨模型术后4周发现β-TCP+siRNA-NLK转染的hBMSCs成骨诱导组较其他3组形成更多矿化组织，并且成骨相关标记物BMP2及成神经相关标记物S100显著高表达。结论: siRNA沉默NLK基因可以促进hBMSCs体外神经向分化，并在裸鼠体内异位成骨模型中能够促进神经化组织工程骨的形成。

关键词: 小干扰RNA, NLK基因, 神经向分化, 骨组织工程

Abstract:

Objective: To identify the role of gene silencing or overexpression of Nemo-like kinase (NLK) during the process of neural differentiation of human mesenchymal stem cells (hBMSCs), and to explore the effect of NLK downregulation by transfection of small interfering RNA (siRNA) on promoting neuralized tissue engineered bone regeneration. Methods: NLK-knockdown hBMSCs were established by transfection of siRNA (the experimental group was transfected with siRNA silencing the NLK gene, the control group was transfected with control siRNA and labeled as negative control group), and NLK-overexpression hBMSCs were established using lentivirus vector transfection technique (the experimental group was infected with lentivirus overexpressing the NLK gene, the control group was infected with an empty vector lentivirus and labeled as the empty vector group). After neurogenic induction, quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression of neural-related gene, and Western blot as well as immunofluorescence staining about several specific neural markers were used to evaluate the neural differentiation ability of hBMSCs.6-week-old male nude mice were divided into 4 groups: ① β-tricalcium phosphate (β-TCP) group, ② β-TCP+ osteogenic induced hBMSCs group, ③ β-TCP+ siRNA-negative control (siRNA-NC) transfection hBMSCs group, ④ β-TCP+ siRNA-NLK transfection hBMSCs group. Four weeks after the subcutaneous ectopic osteogenesis models were established, the osteogenesis and neurogenesis were detected by hematoxylin-eosin (HE) staining, Masson staining and tissue immunofluorescence assay. Statistical analysis was conducted by independent sample t test. Results: After gene silencing of NLK by siRNA in hBMSCs, neural-related genes, including the class Ⅲ β-tubulin (TUBB3), microtubule association protein-2 (MAP2), soluble protein-100 (S100), nestin (NES), NG2 proteoglycan (NG2) and calcitonin gene-related peptide (CGRP), were increased significantly in NLK-knockdown hBMSCs compared with the negative control group(P < 0.05), and the expression levels of TUBB3 and MAP2 of the NLK silencing group were also increased. Oppositely, after NLK was overexpressed using lentivirus vector transfection technique, TUBB3, MAP2, S100 and NG2 were significantly decreased in NLK-overexpression hBMSCs compared with the empty vector group (P < 0.05), and the expression level of TUBB3 was also decreased. 4 weeks after the subcutaneous ectopic osteogenesis model was established, more mineralized tissues were formed in the β-TCP+ siRNA-NLK transfection hBMSCs group compared with the other three groups, and the expression of BMP2 and S100 was higher in the β-TCP+ siRNA-NLK transfection hBMSCs group than in the other groups. Conclusion: Gene silencing of NLK by siRNA promoted the ability of neural differentiation of hBMSCs in vitro and promoted neuralized tissue engineered bone formation in subcutaneous ectopic osteogenic models in vivo in nude mice.

Key words: Small interfering RNA, NLK gene, Neural-differentiation, Bone tissue engineering

中图分类号: 

  • R783.3

表1

实时荧光定量PCR引物序列"

Gene Forward primer (5′-3′) Reverse primer (5′-3′)
GADPH CGACAGTCAGCCGCATCTT CCAATACGACCAAATCCGTTG
NLK CCAACCTCCACACATTGACTATT ACTTTGACATGATCTGAGCTGAG
TUBB3 AGTATCCCGACCGCATCA CATCCGTGTTCTCCACCAG
MAP2 ACCCTCTTCATCCTCCCTGT ACCTCACTGGACCTCAGCAC
NES CTCTGACCTGTCAGAAGAAT CCCACTTTCTTCCTCATCTG
NG2 CACGGCTCTGACCGACATAG CCCAGCCCTCTACGACAGT
CGRP ATGGGCTTCCAAAAGTTCTC TTAGTTGGCATTCTGGGGCATG
S100 TGAAGAAATCCGAACTGAAGGA GAATTCCTGGAAGTCACATTCG

图1

siRNA沉默NLK效率检测及沉默NLK促进hBMSCs成神经向分化的基因、蛋白表达情况"

图2

沉默NLK促进hBMSCs成神经向分化的细胞免疫荧光检测"

图3

过表达NLK效率检测及过表达NLK抑制hBMSCs成神经向分化的基因、蛋白表达情况"

图4

术后4周裸鼠皮下异位成骨标本组织学观察"

图5

术后4周裸鼠皮下异位成骨标本组织切片免疫组织化学染色"

图6

术后4周裸鼠皮下异位成骨标本组织切片免疫荧光染色"

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ISSN 1671-167X
CN 11-4691/R
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