IDH1突变通过影响软骨分化促进软骨肿瘤的发生

IDH1 mutation promotes the development of cartilage tumors by affecting cartilage differentiation

目的:研究IDH1突变在软骨肿瘤发生过程中对软骨分化能力的影响。方法:利用Sanger测序法,检测收集的人软骨肿瘤石蜡标本中IDH1的突变情况;诱导小鼠胚胎间充质干细胞向成骨细胞分化,转染IDH1 R132H位点突变的质粒,观察细胞分化情况;分离培养IDH1突变小鼠的原代软骨细胞,Real-time PCR方法检测软骨分化标志分子Sox9和Col-II以及软骨细胞肥大标志分子Runx2和Col-X基因的表达水平;构建软骨组织特异性突变的IDH1-KI小鼠,采用阿尔新蓝-茜素红全骨架染色,研究IDH1突变如何影响软骨细胞的分化功能。结果:IDH1突变是软骨肿瘤中常见的遗传学改变;IDH1 R132H位点突变之后,细胞分泌的2-HG浓度显著增高;加入成骨细胞分化诱导液之后,胚胎间充质干细胞能够向成骨细胞分化,而当IDH1 R132H位点突变后,干细胞无法正常分化;IDH1突变小鼠的软骨细胞中,软骨分化标志性分子Sox9和Col-II基因表达无差异,然而软骨细胞肥大标志性分子Runx2和Col-X基因表达显著高于野生组小鼠,利用5 mmol/L的2-HG刺激模拟IDH1突变,得到相似结果;IDH1-KI小鼠整个骨骼中软骨含量明显增多,且有明显的骨化异常。结论:IDH1突变促进了软骨细胞的生长,抑制了软骨到成骨的分化过程。

Objective:To investigate the effect of IDH1 mutation on the cartilage differentiation during cartilage tumors tumorigenesis.Methods:IDH1 mutations in paraffin specimens of human cartilage tumors were detected by Sanger sequencing method.Mouse embryonic mesenchymal stem cells were induced to differentiate into osteoblasts and transfected with IDH1-R132H mutant plasmid to observe cell differentiation.Primary chondrocytes of IDH1 mutant mice were isolated and cultured.The expression levels of Sox9 and Col-II markers of chondrogenic differentiation and Runx2 and Col-X markers of chondrocyte hypertrophy were detected by Real-time PCR.We generated conditional IDH1-KI mice,and whole-mount skeletal staining with alcian blue and alizarin red was used to determine how mutant IDH1 altered chondrocyte function in vivo.Results:Human cartilage tumors have a broad variety of IDH1mutations.After IDH1-R132H mutation,the concentration of 2-HG secreted by cells increased significantly.Embryonic mesenchymal stem cells could differentiate into osteoblasts after the addition of osteoblast differentiation induction solution,but could not differentiate normally after IDH1-R132H mutation.In the chondrocytes of IDH1 mutant mice,there was no difference in the expression of Sox9 and Col-II genes which are the markers of chondrocyte differentiation.However,the expression of Runx2 and Col-X genes,the markers of chondrocyte hypertrophy,was significantly higher than that of wild mice.Treatment of WT cells with 2-HG has a similar effect on gene expression as observed in cells expressing IDH1-R132H.The cartilage content in the whole bones of IDH1-KI mice increased significantly,and there was obvious disfunction of osteoblast differentiation.Conclusion:IDH1 mutations promote the growth of chondrocytes and inhibit the differentiation process from cartilage to formation.