人脐带间充质干细胞通过Wnt/β-连环蛋白信号通路防治大鼠激素性骨质疏松  

Effect of human umbilical cord mesenchymal stem cells on glucocorticoid-induced osteoporosis in rats through Wnt/β-catenin signaling pathway

作　　者：王学中 邓爽[1] 张宇标 刘子林 黄俊[1] 彭昊[1] Wang Xuezhong;Deng Shuang;Zhang Yubiao;Liu Zilin;Huang Jun;Peng Hao(Department of Orthopedics,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院骨科,430060

出　　处：《中华实验外科杂志》2022年第7期1247-1251,共5页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金(040010002)。

摘　　要：目的探讨人脐带间充质干细胞(HUC-MSCs)在防治大鼠激素性骨质疏松(GIOP)方面的疗效及其潜在的分子生物学机制。方法采用光学显微镜、流式细胞技术对HUC-MSCs进行表征和鉴定。成骨细胞MC3T3-E1分为对照组、地塞米松(DEX)组、DEX+不同比例HUC-MSCs组。对照组加入正常培养基,DEX组加入10μmol/L DEX处理,DEX+不同比例HUC-MSCs组加入10μmol/L DEX处理细胞,然后将HUC-MSCs按照1∶1、10∶1、100∶1的比例在共培养小室中与MC3T3-E1非接触式共培养48 h。采用细胞计数试剂盒(CCK-8)法检测细胞活力。24只SD大鼠随机分成3组(n=8):对照组、模型组、治疗组(HUC-MSCs组)。模型组使用DEX构建GIOP模型,对照组给予等量磷酸盐缓冲液(PBS),治疗组在构建GIOP模型的同时通过尾静脉注射约106个HUC-MSCs进行治疗。8周后取股骨组织样本,采用苏木精-伊红(HE)染色观察空骨陷窝率,脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)染色评价细胞凋亡。进行Micro CT扫描,测量骨密度(BMD)、单位组织体积的骨量(Bv/Tv)、骨小梁厚度(Tb.Th)、骨小梁间距(Tb.Sp)、骨小梁数量(Tb.N)等骨质疏松相关指标。提取组织蛋白后,用蛋白印迹法(Western blot)检测通路蛋白表达。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果流式细胞鉴定结果显示,HUC-MSCs高表达CD44(99.9%)、CD73(98.0%)、CD90(100.0%)、CD105(99.6%)、CD166(99.9%),低表达CD14(0.43%)、CD19(1.11%)、CD34(0.89%)、CD45(1.06%)、HLA-DR(0.38%),符合HUC-MSCs表面特异性抗原表达规律。CCK-8显示,模型组较对照组细胞活力明显下降,与HUC-MSCs共培养后可部分恢复MC3T3-E1活力。HE染色对照组、模型组、HUC-MSCs组空骨陷窝率分别为(2.546±1.049)%、(28.720±1.546)%、(13.600±1.012)%。TUNEL染色结果对照组、模型组、HUC-MSCs组TUNEL阳性细胞的比例分别为(2.334±0.789)%、(24.140±4.646)%、(11.610±2.974)%。Micro CT显示模型组BMObjective To investigate the effect and potential molecular biological mechanism of human umbilical cord mesenchymal stem cells(HUC-MSCs)on glucocorticoid-induced osteoporosis(GIOP)in rats.Methods HUC-MSCs were characterized and identified by optical microscopy and flow cytometry.Osteoblasts MC3T3-E1 were divided into control group,dexamethasone(DEX)group,DEX+HUC-MSCs group(the ratio of HUC-MSCs and MC3T3-E1:1∶1,10∶1 and 100∶1 respectively).Normal medium was added to control group.DEX group was treated with 10μmol/L DEX.In the DEX+HUC-MSCs group,osteoblasts MC3T3-E1 were treated with 10μmol/L DEX,HUC-MSCs were then non-contact co-cultured with MC3T3-E1 in co-culture chamber in a ratio of 1∶1,10∶1,and 100∶1 respectively for 48 h.Cell viability was detected by cell counting kit-8(CCK-8).A total of 24 SD rats were randomly divided into 3 groups(n=8):control group,model group and treatment group(HUC-MSCs group).The GIOP model was constructed by DEX administration,and the control group was given the same volume of phosphate buffer saline(PBS).The HUC-MSCs group was treated with about 106 HUC-MSCs through tail vein during the construction of GIOP model.At 8th week,femur tissue samples were collected.Hematoxylin-eosin(HE)staining was employed to measure the rate of empty lacunae,and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining was used to evaluate osteoblast apoptosis.Micro-CT scanning was performed to measure the indicators related to osteoporosis,including bone mineral density(BMD),bone volume per tissue volume(Bv/Tv),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp),trabecular number(Tb.N).Total tissue proteins were extracted according to the manufacturer’s protocol,and the relative expression of pathway proteins was detected by Western blotting.Results Flow cytometry showed that HUC-MSCs highly expressed CD44(99.9%),CD73(98.0%),CD90(100.0%),CD105(99.6%),and CD166(99.9%),while down-regulated CD14(0.43%),CD19(1.11%),CD34(0.89%),CD45(1.06%)and HLA-DR(0.38%),whic

关 键 词：人脐带间充质干细胞 Wnt/β-连环蛋白信号通路 骨质疏松 Β-连环蛋白 骨形态发生蛋白-2 

分 类 号：R580[医药卫生—内分泌]