敲低NIPBL基因调控小鼠骨髓间充质干细胞向软骨的分化  

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

作　　者：马雯晴 张惠荣[2] 刘辉 董丽丽 杨眷娣 Ma Wenqing;Zhang Huirong;Liu Hui;Dong Lili;Yang Juandi(Medical School of Shihezi University,Shihezi 832003,Xinjiang Uygur Autonomous Region,China;Department of Pediatrics,First Affiliated Hospital of Medical School of Shihezi University,Shihezi 832003,Xinjiang Uygur Autonomous Region,China)

机构地区：[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

出　　处：《中国组织工程研究》2023年第10期1477-1483,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81660260),项目负责人:张惠荣。

摘　　要：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 sh RNA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 m RNA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。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 sh RNA 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 m RNA 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 tha

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 

分 类 号：R446[医药卫生—诊断学] R318[医药卫生—临床医学] R729