体外培养GFP裸小鼠颅骨细胞的表形分析

Morphological analysis of GFP nude mice skull cells cultured in vitro

目的本研究建立的转绿色荧光蛋白基因近交系裸小鼠(GFP裸小鼠)已在示踪研究胶质瘤微环境方面起了重要作用,但在治疗自体颅骨成形术后骨吸收并发症的效果及机制方面未见报告,本文旨在分析体外培养GFP裸小鼠颅骨细胞的表形,为治疗颅骨吸收准备工具细胞奠定基础。方法取生后3 d的GFP裸小鼠,在无菌条件下解剖双侧顶骨,连同骨膜,将颅骨剪成1 mm2左右的小片,置于含有胎牛血清的1640培养基中,在5%CO2培养箱中作短期传代培养,收集从骨片上长出的细胞作相关检测。结果对原代(P0)和继代(P1、P2)细胞观察表明,在60 mm皿底长满90%的传代时间约6~8 d,抽样细胞计数约2.3×106~2.5×106个/皿,形态以纤维形为主,也有星形和树突状;在荧光显微镜下所有细胞全部发绿色荧光,形态与白光镜下一致;标志蛋白检测表明,在整个细胞群中同时存在BMP-6+的成骨祖细胞和CD206+、CD68+的巨噬细胞。结论基于颅骨再生,除了成骨祖细胞作为起始细胞,还必须有巨噬细胞参与维持环境稳态,培养成功的P0,P1和P2三代细胞因同时满足这个需要,有望作为工具细胞进一步用于颅骨再生的研究。

Objective The inbred transgenic nude mice with green fluorescent protein gene(GFP nude mice)line that we established have played an important role in tracing studies of the glioma microenvironment,but no study has yet reported its mechanism and effect in treating bone resorption complications after autologous cranioplasty.Our purpose in this article is to analyze the phenotype of GFP nude mouse skull cells cultured in vitro to lay the foundation for preparing tool cells for the treatment of skull resorption.Methods We selected GFP mice within three days after birth.Under sterile conditions,bilateral parietal bones with periosteum were isolated and cut into small 1 mm2 pieces,and then placed in 1640 medium with fetal bovine serum.We then incubated the samples in a 5%CO2 incubator.Those cells that crawled out of the bone slices were collected for short-term subculture and relevant analyses.Results Observation of primary(P0)and secondary(P1,P2)cells indicates that the passage time in which cells grow to cover 90%of the bottom of a 60 mm dish is about 6~8 days,with a sampling calculation of 2.3×106~2.5×106 cells per dish.The cell morphology is mainly fibrous,but also star-shaped and dendritic.All cells emit green fluorescence under a fluorescent microscope,which is consistent with that under a white light microscope.Marker protein detection showed that BMP-6+osteoblasts and CD206+,CD68+macrophages coexist in the entire cell population.Conclusions Based on our skull regeneration studies,macrophages must also be involved in maintaining environmental homeostasis,in addition to the osteoblastic progenitor cell role as a starting cell.Successfully cultured P0,P1,and P2 third-generation cells meet this need,and are all expected to be further used as a tool cell in skull regeneration research.