Gene expression in alpha SMA Cre-labeled cells in periosteum during early fracture callus formation

Fracture healing is a process that involves many cell populations. In this study we characterized gene expression in a subset of cells involved in fracture healing. αSMACreERT2 mice crossed with Ai9 reporter mice that express tdTomato fluorescent protein after Cre-mediated activation were used as an experimental model. αSMA-expressing cells were labeled by tamoxifen administration, then periosteal cells from the tibia were isolated two days later (controls), or tibial fractures were performed and periosteum/soft callus tissue was collected after 2 and 6 days. The tdTomato positive cell population was isolated by flow cytometry, and subjected to microarray analysis. Histology and cell surface marker analysis indicates that αSMACreERT2 labels a mainly mesenchymal population in the periosteum that expands after fracture, and contributes to both osteogenic and chondrogenic elements of the fracture callus. We were therefore able to examine gene expression in a defined population during the early stages of fracture healing. Total RNA was obtained from the tomato positive cells within the periosteal compartment of fractures from αSMACreERT2/Ai9 mice. Control animals were given 2 doses of tamoxifen, and periosteum was collected and labeled cells sorted (8-9 sex-matched mice per group). Fractures were performed after the second dose of tamoxifen, and tomato positive cells from periosteum/callus tissue were isolated 2 and 6 days after fracture (4-8 animals per sample pooled). 3 replicates for each sample are included.

骨折愈合是一个涉及多种细胞群的复杂生物学过程。本研究针对参与骨折愈合的特定细胞亚群，系统表征其基因表达特征。本研究采用的实验模型为：将αSMACreERT2小鼠与Ai9报告小鼠（Cre介导激活后可表达tdTomato荧光蛋白）进行杂交。通过他莫昔芬给药对表达α平滑肌肌动蛋白（αSMA）的细胞进行标记：对照组于给药2天后分离胫骨骨膜细胞；实验组则构建胫骨骨折模型，分别于骨折后2天和6天收集骨膜/纤维骨痂组织。通过流式细胞术分选出tdTomato阳性细胞群，并对其进行基因芯片（microarray）分析。组织学与细胞表面标志物分析结果表明：αSMACreERT2可标记骨膜内以间充质细胞为主的细胞群，该细胞群在骨折后发生扩增，并参与构成骨折骨痂的成骨与成软骨组分。基于上述结果，本研究得以在骨折愈合早期阶段，对特定细胞群的基因表达进行精准分析。本研究从αSMACreERT2/Ai9杂交小鼠骨折部位骨膜区的tdTomato阳性细胞中提取总RNA：对照组小鼠给予2次他莫昔芬给药，随后收集骨膜并分选出标记阳性细胞（每组使用8-9只性别匹配的小鼠）；实验组于第二次他莫昔芬给药后构建胫骨骨折模型，分别于骨折后2天和6天收集骨膜/骨痂组织并分选出tdTomato阳性细胞（每份样本混合4-8只小鼠的组织），每个样本设置3次生物学重复。