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

Q3 Medicine
北京大学学报(医学版) Pub Date : 2025-04-18
Mengdi Li, Lei Lei, Zhongning Liu, Jian Li, Ting Jiang
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引用次数: 0

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.

[nemo样激酶基因沉默促进神经化组织工程骨再生]。
目的:探讨nemo样激酶(NLK)基因沉默或过表达在人间充质干细胞(hBMSCs)神经分化过程中的作用,并探讨转染小干扰RNA (siRNA)下调NLK对神经化组织工程骨再生的促进作用。方法:通过转染siRNA(实验组转染沉默NLK基因的siRNA,对照组转染对照siRNA,标记为阴性对照组)建立NLK低表达的hBMSCs,采用慢病毒载体转染技术(实验组转染过表达NLK基因的慢病毒)建立NLK过表达的hBMSCs。对照组用空载体慢病毒感染,标记为空载体组。神经原性诱导后,采用定量实时聚合酶链反应(qPCR)检测神经相关基因的表达,并采用Western blot和几种特异性神经标志物的免疫荧光染色评价hBMSCs的神经分化能力。将6周龄雄性裸鼠分为4组:①β-磷酸三钙(β-TCP)组,②β-TCP+成骨诱导hBMSCs组,③β-TCP+ sirna阴性对照(siRNA-NC)转染hBMSCs组,④β-TCP+ siRNA-NLK转染hBMSCs组。建立皮下异位成骨模型4周后,采用苏木精-伊红(HE)染色、Masson染色和组织免疫荧光法检测成骨和神经发生情况。统计学分析采用独立样本t检验。结果:siRNA沉默NLK基因后,与阴性对照组相比,NLK敲除hBMSCs中Ⅲβ-微管蛋白(TUBB3)、微管关联蛋白-2 (MAP2)、可溶性蛋白-100 (S100)、巢蛋白(NES)、NG2蛋白多糖(NG2)、降钙素基因相关肽(CGRP)类神经相关基因显著升高(P < 0.05), NLK沉默组TUBB3、MAP2表达水平也升高。相反,慢病毒载体转染NLK过表达后,NLK过表达的hBMSCs中TUBB3、MAP2、S100和NG2较空载体组显著降低(P < 0.05), TUBB3的表达水平也降低。皮下异位成骨模型建立4周后,β-TCP+ siRNA-NLK转染hBMSCs组较其他3组形成更多矿化组织,且β-TCP+ siRNA-NLK转染hBMSCs组BMP2和S100表达高于其他3组。结论:siRNA沉默NLK基因可促进hBMSCs体外神经分化能力,促进裸鼠体内皮下异位成骨模型的神经化组织工程骨形成。
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来源期刊
北京大学学报(医学版)
北京大学学报(医学版) Medicine-Medicine (all)
CiteScore
0.80
自引率
0.00%
发文量
9815
期刊介绍: Beijing Da Xue Xue Bao Yi Xue Ban / Journal of Peking University (Health Sciences), established in 1959, is a national academic journal sponsored by Peking University, and its former name is Journal of Beijing Medical University. The coverage of the Journal includes basic medical sciences, clinical medicine, oral medicine, surgery, public health and epidemiology, pharmacology and pharmacy. Over the last few years, the Journal has published articles and reports covering major topics in the different special issues (e.g. research on disease genome, theory of drug withdrawal, mechanism and prevention of cardiovascular and cerebrovascular diseases, stomatology, orthopaedic, public health, urology and reproductive medicine). All the topics involve latest advances in medical sciences, hot topics in specific specialties, and prevention and treatment of major diseases. The Journal has been indexed and abstracted by PubMed Central (PMC), MEDLINE/PubMed, EBSCO, Embase, Scopus, Chemical Abstracts (CA), Western Pacific Region Index Medicus (WPR), JSTChina, and almost all the Chinese sciences and technical index systems, including Chinese Science and Technology Paper Citation Database (CSTPCD), Chinese Science Citation Database (CSCD), China BioMedical Bibliographic Database (CBM), CMCI, Chinese Biological Abstracts, China National Academic Magazine Data-Base (CNKI), Wanfang Data (ChinaInfo), etc.
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