构建髓系细胞特异性Clcn7突变骨硬化症小鼠模型和表型研究

Generation and phenotype of myeloid cell specific Clcn7-mutation osteopetrosis mouse model

目的构建髓系细胞特异性Clcn7-G763R突变的骨硬化小鼠模型,并进行表型研究。方法构建条件性敲入Clcn7基因p.G763R错义突变小鼠,并与溶菌酶M启动子-重组酶转基因小鼠(LysM cre)交配繁殖,最终获取髓系细胞特异性Clcn7-G763R突变的骨硬化小鼠。利用Micro CT分析不同基因型小鼠骨微细结构的差异,利用HE染色观察骨组织形态学改变。诱导原代破骨细胞,通过实时荧光定量PCR技术检测破骨细胞分化和成熟相关基因表达水平。利用骨吸收陷窝实验观察破骨细胞功能。结果4周龄时纯合突变小鼠体重较野生型小鼠减轻[(12.000±1.666)g对(15.630±2.314)g,P=0.021],股骨长度较野生型缩短[(1.160±0.096)cm对(1.300±0.082)cm,P=0.037)]。Micro CT发现纯合突变小鼠骨密度在4周龄时较野生型小鼠显著增加[(0.753±0.002)g/cm^(3)对(0.143±0.034)g/cm^(2),P=0.003],而杂合突变小鼠骨密度在8周龄时增加明显[(0.236±0.021)g/cm^(3)对(0.180±0.020)g/cm^(3),P=0.030]。HE染色发现纯合突变小鼠骨髓腔消失,松质骨骨小梁数量显著增多,软骨肥大区增宽;杂合突变小鼠骨小梁较野生型小鼠增多。体外实验中,杂合突变小鼠破骨细胞数量较野生型无明显变化(P=0.358),但破骨细胞面积增大[(3.590×10^(6)±0.911×10^(6))μm^(2)对(1.352×10^(6)±0.260×10^(6))μm^(2),P=0.043],骨吸收陷窝相关检测结果提示破骨细胞功能降低,而破骨细胞分化和成熟相关基因NFATc1、c-fos、Ctsk和Acp5表达水平均无显著变化。结论本研究成功构建髓系细胞特异性Clcn7-G763R突变的小鼠,突变型小鼠破骨细胞功能受损,骨量显著增加且股骨变短,为后续机制研究和治疗探索提供工具。

Objective To construct a myeloid cell specific Clcn7-G763R mutant mouse model and characterize its phenotype.Methods A mouse conditional knocked in p.G763R mutation in Clcn7 gene was constructed and bred with LysM cre mice to obtain osteopetrosis mice with myeloid cell specific Clcn7-G763R mutation.The differences of bone mass in mice with different genotypes were analyzed using Micro CT and the changes of histology were observed with HE staining.Osteoclasts were cultured and the expression levels of osteoclasts differentiation and maturation-related genes were detected by real-time PCR.The functions of osteoclasts were examined through bone resorption assay.Results The body weight of homozygous mutant mice at 4 weeks old was reduced compared with the wild type mice[(12.000±1.666)g vs(15.630±2.314)g,P=0.021],with shorter femur length[(1.160±0.096)cm vs(1.300±0.082)cm,P=0.037].Micro CT showed that bone mineral density of homozygous mutant mice was remarkably increased at 4 weeks old[(0.753±0.002)g/cm^(3)vs(0.143±0.034)g/cm^(3),P=0.003],while bone mineral density of heterozygous mutant mice increased significantly at 8 weeks old[(0.236±0.021)g/cm^(3)vs(0.180±0.020)g/cm^(3),P=0.030].HE staining revealed increased trabecula bone volume in the mutant mice,especially in homozygous mutant mice with narrow bone marrow cavity and wider hypertrophic zone of chondrocytes.There was no significant difference in the number of osteoclasts between wild type mice and heterozygous mice in vitro(P=0.358),while total area of osteoclasts increased in heterozygous mutant mice[(3.590×10^(6)±0.911×10^(6))μm^(2)vs(1.352×10^(6)±0.260×10^(6))μm^(2),P=0.043].Impaired function of resorption was unveiled by bone resorption assay.There were no significant differences in the expressions of osteoclast differentiation and maturity-related genes including NFATc1,c-fos,Ctsk,and Acp5 between the two groups.Conclusion A myeloid cell specific Clcn7-G763R mutation mice with impaired osteoclasts and increased bone mass is successfully constructed.