股骨干骨折后过度生长动物模型制作及差异蛋白质组学研究

Animal model and analysis of differential proteomics in overgrowth after femoral shaft fractures

目的建立股骨干骨折后过度生长动物模型,并通过蛋白质组学研究观察骺板的差异表达蛋白,以期明确股骨干骨折后过度生长的生物学机制。方法取48只雄性6周龄日本大耳白兔,利用环形剥离股骨干骨膜的方法建立兔股骨干骨折后过度生长动物模型,右侧为手术侧,左侧为假手术侧。术后4周取术侧及假手术侧股骨测量全长,并取股骨远端骺板行串联质谱标签(tandem mass tag,TMT)定量蛋白质组学分析,通过对差异蛋白行基因本体(gene ontology,GO)分析及京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析研究相关蛋白表达变化。另取20只10周龄雄性日本大耳白兔作为空白对照组,测量其双侧股骨长度。对相关差异蛋白行蛋白质印迹法检测。股骨长度差及蛋白表达差异采用Kolmogorov-Smirnov检验验证正态性,F检验验证方差齐性,符合正态分布且方差齐,采用配对t检验比较分析两侧股骨长度,独立样本t检验比较分析手术组与空白组长度差,若不符合正态分布或方差不齐采用秩和检验。GO功能富集分析应用Fisher精确检验。结果术后4周,手术组双侧股骨长度差(手术侧-假手术侧)为(0.62±0.63)mm,空白组双侧股骨长度差(右侧-左侧)为(-0.025±0.26)mm,组间比较,差异有统计学意义(P<0.001)。共鉴定出283种差异蛋白,GO分析提示差异蛋白富集于细胞代谢、胞内结构、有机环状化合物结合等,KEGG富集提示差异蛋白通路集中于核糖体、蛋白质外排、剪切体、细胞凋亡及内质网中蛋白质加工等。蛋白质印迹法检测提示X型胶原蛋白α1链(type X collagen alpha 1 chain,COL10A1)在过度生长的股骨远端骺板软骨中表达上调。结论通过骨膜环切法可建立股骨过度生长的动物模型,COL10A1可能参与股骨过度生长的生物学行为。

Objective To establish an animal model in overgrowth after femoral shaft fracture and screen for differentially expressed proteins in growth plate via differential proteomics so as to verify the biological mechanisms of this phenomenon.Methods Femoral diaphyseal periosteum stripping was performed as an animal model in overgrowth after fractures on 48 six-week-old male Japanese white rabbits.Periosteum was stripped on right sides with sham operation at left sides.Full femoral lengths were measured at Week 4 post-operation with distal growth plates taken for TMT quantified proteomic analysis.Differential protein expressions were analyzed via Gene Ontology analysis(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Twenty other 10-week-old male rabbits were taken as blank control group and femoral full lengths measured.Part of the results was validated by Western blot by measuring the expression levels of COL10A1 between operation and sham-operation sides.Results Leg length discrepancy in operation group(operation side minus sham-operation side)was(0.62±0.63)mm longer than that in control group(right side minus left side,(-0.025±0.26)mm,P<0.001).A total of 283 proteins with significant differences were identified(P<0.05).GO analysis revealed that different proteins were principally involved in cellular metabolic process,intracellular components and organic cyclic compound bonding,etc.Significant signaling pathways were detected by KEGG analysis.Pathways were correlated with ribosome,protein export,spliceosome,necroptosis and protein processing in endoplasmic reticulum.Col10al(typeⅩcollagen alpha 1)was up-regulated in distal femoral growth plate at operated side.Conclusions Overgrowth after fractures may be established by periosteum stripping in rabbits.And COL10A1 may participate in the biological behaviors of overgrowth after femoral shaft fractures.