中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (10): 1477-1483.doi: 10.12307/2023.288

• 骨髓干细胞 bone marrow stem cells •    下一篇

敲低NIPBL基因调控小鼠骨髓间充质干细胞向软骨的分化

马雯晴1,张惠荣2,刘  辉1,董丽丽1,杨眷娣1   

  1. 1石河子大学医学院,新疆维吾尔自治区石河子市   832003;2石河子大学医学院第一附属医院儿科,新疆维吾尔自治区石河子市   832003
  • 收稿日期:2022-04-03 接受日期:2022-05-24 出版日期:2023-04-08 发布日期:2022-09-06
  • 通讯作者: 张惠荣,主任医师,硕士生导师,石河子大学医学院第一附属医院儿科,新疆维吾尔自治区石河子市 832003
  • 作者简介:马雯晴,女,1996年生,山东省淄博市人,汉族,石河子大学医学院在读硕士,主要从事新生儿疾病方面的研究。
  • 基金资助:
    国家自然科学基金项目(81660260),项目负责人:张惠荣

Knockdown of NIPBL gene regulates chondrogenic differentiation of mouse bone marrow mesenchymal stem cells

Ma Wenqing1, Zhang Huirong2, Liu Hui1, Dong Lili1, Yang Juandi1   

  1. 1Medical School of Shihezi University, Shihezi 832003, Xinjiang Uygur Autonomous Region, China; 2Department of Pediatrics, First Affiliated Hospital of Medical School of Shihezi University, Shihezi 832003, Xinjiang Uygur Autonomous Region, China
  • Received:2022-04-03 Accepted:2022-05-24 Online:2023-04-08 Published:2022-09-06
  • Contact: Zhang Huirong, Chief physician, Master’s supervisor, Department of Pediatrics, First Affiliated Hospital of Medical School of Shihezi University, Shihezi 832003, Xinjiang Uygur Autonomous Region, China
  • About author:Ma Wenqing, Master candidate, Medical School of Shihezi University, Shihezi 832003, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81660260 (to ZHR)

摘要:

文题释义:
Cornelia de Lange综合征:是一种由基因突变引起的多器官系统发育异常综合征,临床患儿主要表现为身体智力发育迟缓、认知障碍、面容畸形、肢体骨骼发育缺陷等。该病由NIPBL、MAU2、SMC1A、SMC3、RAD21、BRD4、HDAC8、ANKRD11、EP300基因的致病性变异所致,其中NIPBL占60%,且该病80%最严重的临床表现涉及NIPBL突变。
NIPBL基因:是位于5号染色体的类似于B蛋白的基因(Nipped-B-like protein),其编码的Delangin蛋白在胚胎和成人心脏、骨骼肌及脑等组织中大量表达,具有调节胚胎时期器官组织发育的作用。同时,在染色单体凝聚、基因表达和DNA损伤修复中发挥重要作用。

背景:目前已将NIPBL基因突变作为诊断Cornelia de Lange综合征的首选指标,但由于该病的遗传异质性,显著增加了临床诊疗难度,尤其患儿骨骼发育畸形的发生率高,其发病机制尚不明确,目前无有效治疗方案,患儿平均寿命较一般人群显著缩短。
目的:探究敲低NIPBL基因对小鼠骨髓间充质干细胞成软骨分化能力的影响及其可能的分子调控机制。
方法:将慢病毒转染NIPBL shRNA的小鼠骨髓间充质干细胞作为sh-NIPBL组,慢病毒空载体转染的骨髓间充质干细胞作为sh-NC组,无慢病毒干扰的骨髓间充质干细胞作为空白对照组,对上述3组细胞进行成软骨诱导培养,诱导21 d后测量软骨微球最大横截面的周长,行阿利辛蓝染色鉴定其诱导分化结果,免疫荧光法检测软骨细胞Ⅱ型胶原的表达水平,应用实时荧光定量PCR法检测软骨细胞Sox-9、TGF-β1、Smad2、Smad4 mRNA表达水平。
结果与结论:①成软骨诱导第21天,sh-NIPBL组的软骨微球最大横截面的周长明显小于对照组(P < 0.05),阿利辛蓝染色后在倒置显微镜下观察sh-NC组较sh-NIPBL组可见更多的蓝色软骨内酸性黏多糖;②诱导成软骨分化后,sh-NIPBL组骨髓间充质干细胞中Ⅱ型胶原、Sox-9的表达低于sh-NC组和空白对照组(P < 0.05);③成软骨诱导过程中,sh-NIPBL组TGF-β1、Smad2、Smad4基因的表达水平低于sh-NC组和空白对照组(P < 0.05);④结果表明,慢病毒敲低NIPBL基因表达降低了骨髓间充质干细胞的成软骨分化能力,且该过程可能由TGF-β1/Smad信号通路参与介导。

https://orcid.org/0000-0002-4727-6528 (马雯晴)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: NIPBL基因, 骨骼发育缺陷, 慢病毒转染, 成软骨分化, TGF-β1/Smad信号通路, Cornelia de Lange 综合征

Abstract: BACKGROUND: At present, NIPBL gene mutation has been used as the preferred indicator for the diagnosis of Cornelia de Lange syndrome. However, due to the genetic heterogeneity of the disease, clinical diagnosis and treatment are more difficult; especially, the incidence of skeletal dysplasia in children is high, and its pathogenesis is still unclear. There is currently no targeted therapy program, and the average life expectancy of children is significantly shorter than that of the general population.  
OBJECTIVE: To investigate the effect of knockdown of NIPBL gene on chondrogenic differentiation ability of mouse bone marrow mesenchymal stem cells and its possible molecular regulation mechanism.
METHODS:  Mouse bone marrow mesenchymal stem cells transfected with NIPBL shRNA by lentivirus were used as sh-NIPBL group, and bone marrow mesenchymal stem cells transfected with lentivirus empty vector were used as sh-NC group. Bone marrow mesenchymal stem cells without lentivirus interference were used as the blank control group. Next, the chondrogenic induction culture was carried out in the three groups. After 21 days of induction, the perimeter of the largest cross section of cartilage microspheres was measured and Alicia blue staining was used to identify the induced differentiation. The expression level of Collagen II was detected by immunofluorescence method. The expression levels of Sox-9, TGF-β1, Smad2, and Smad4 mRNA in chondrocytes were detected by real-time quantitative PCR.
RESULTS AND CONCLUSION: (1) On day 21 of chondrogenic induction, the perimeter of the largest cross section of cartilage microspheres in the sh-NIPBL group was significantly smaller than that in the control group (P < 0.05). More blue intrachondral acidic mucopolysaccharides were observed in the sh-NC group than that in the sh-NIPBL group after alician blue staining under an inverted microscope. (2) After induction into chondrogenic differentiation, the expression levels of Collagen II and Sox-9 in bone marrow mesenchymal stem cells of the sh-NIPBL group were lower than those in the sh-NC and blank control groups (P < 0.05). (3) In the process of chondrogenic induction, the expression levels of TGF-β1, Smad2, and Smad4 mRNA of the sh-NIPBL group were lower than those in the sh-NC and blank control groups (P < 0.05). (4) These findings suggest that lentiviral knockdown of NIPBL gene expression reduced the chondrogenic differentiation of bone marrow mesenchymal stem cells, and this process was mediated by the TGF-β1/Smad signaling pathway.

Key words: NIPBL gene, skeletal developmental defect, lentiviral transfection, chondrogenic differentiation, TGF-β1/Smad signaling pathway, Cornelia de Lange syndrome

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