大鼠硬脑膜对颅骨成骨增强的影响

Effect of dura mater on enhancement of cranial osteogenesis in rats

背景:硬脑膜与颅骨在结构和功能上联系密切,原代提取硬脑膜和颅骨细胞并将二者共培养的研究几乎没有,利用原代细胞探究硬脑膜对颅骨的影响具有创新性,有望为临床治疗提供理论依据。目的:原代提取大鼠硬脑膜和颅骨细胞,观察硬脑膜对颅骨增殖和分化能力的影响,初步了解Twist1在其中的作用。方法:利用酶解法与组织块法相结合的方法原代提取出生3 d内大鼠硬脑膜细胞和颅骨细胞,免疫荧光染色鉴定所提取细胞,茜素红染色鉴定与评估颅骨细胞及其矿化能力,real-time PCR检测硬脑膜细胞与颅骨细胞共培养后,颅骨细胞增殖与成骨相关基因表达及Twist1的表达。结果与结论:①形态学:所提取硬脑膜细胞形态特征与成纤维细胞一致,成骨细胞呈纺锤形。②细胞鉴定:免疫荧光染色显示,所提取硬脑膜细胞表达高水平的波形蛋白,颅骨细胞表达高水平的碱性磷酸酶;成骨诱导28 d颅骨细胞茜素红染色观察到明显的矿化结节。③real-time PCR检测显示,与对照组比较,共培养组PCNA、碱性磷酸酶、RUNX2 mRNA表达升高(P<0.01);Twist1 mRNA表达降低(P<0.01)。④结果表明,原代提取的颅骨细胞具有较强的矿化能力,硬脑膜是促进颅骨的生长发育与成骨分化的重要因素,且Twist1在该过程中发挥重要作用。

BACKGROUND:The dura mater and skull are physically and functionally related,although there have been few investigations on primary extraction of dura mater and cranial cells,as well as co-culture of the two.The use of primary cells to investigate the influence of the dura mater on the skull is novel,and it is hoped that it may give a theoretical foundation for therapeutic therapy.OBJECTIVE:Rat dura mater and cranial bone cells were retrieved in situ to observe the influence of dura mater on cranial bone proliferation and differentiation,as well as to get a basic knowledge of the involvement of Twist1 in this process.METHODS:The enzyme digestion method was used in conjunction with the tissue block method to extract dural cells and cranial osteoblasts from rats within three days of birth.Immunofluorescence staining was used to identify the extracted cells,and alizarin red staining was used to identify and evaluate cranial osteoblasts and their mineralization ability.After co-culturing dural cells and cranial osteoblasts,real-time PCR was utilized to identify the expression of genes associated to cranial osteoblast proliferation and osteogenesis,as well as Twist1.RESULTS AND CONCLUSION:(1)Morphology:The retrieved dural cells had morphological traits similar to fibroblasts,while the osteoblasts were spindleshaped.(2)Cell identification:immunofluorescence staining revealed that extracted dural cells expressed high levels of vimentin and cranial osteoblasts expressed high levels of alkaline phosphatase;cranial osteoblasts were stained with alizarin red 28 days after osteogenic induction,and obvious mineralized nodules were observed.(3)Real time PCR detection showed that the co-culture group had higher levels of PCNA,alkaline phosphatase,and RUNX2 mRNA expression than the control group(P<0.01);however,Twist1 mRNA expression was lower(P<0.01).(4)The findings showed that the primary extracted cranial osteoblasts had a high mineralization capacity,and that the dura mater was a key factor in promoting cranial growth and development and osteogenic differentiation,with Twist1 playing a key role in this process.