摘要
目的:观察烧伤大鼠血清对体外培养的大鼠骨髓间充质干细胞基因表达的影响。方法:实验于2003-06/2004-4在解放军总医院第一附属医院全军创伤修复重点实验室完成。①断头法处死Wistar大鼠,取股骨并暴露骨髓腔,用培养基冲洗骨髓腔,将骨髓细胞悬液用密度梯度离心法分离骨髓单个核细胞并接种于培养瓶,贴壁法培养骨髓间充质干细胞。②用沸水烫伤的方法制作15%TBSAⅢ度烧伤模型。③烧伤3d后经腹主动脉取烧伤大鼠血液,分离血清备用。④同样取正常大鼠血液,分离血清备用。⑤骨髓间充质干细胞传代后分别用含有体积分数为0.1的正常大鼠血清(正常血清组)和烧伤后3d大鼠血清(烧伤血清组)的F-12培养基培养24h及72h。⑥从处理过的间充质干细胞内提取RNA,反转录为cDNA并以cy3-dUTP和cy5-dUT进行标记为荧光探针。⑦以含有5705种基因的寡核苷酸微阵列检测两组细胞基因表达的差异。结果:①烧伤血清处理不同时间后,两组间充质干细胞对比,均有不同数量的基因表达存在明显差异。②烧伤血清处理24h后,两组相比较有4条基因表达存在明显差异。③烧伤血清处理72h后,两组相比较有11条基因表达存在明显差异。④两个时间点差异表达的基因无相同基因。结论:烧伤后大鼠血清刺激间充质干细胞,使其基因表达发生了改变,且随着烧伤血清处理时间的不同,差异表达的基因也发生动态变化。这种改变可能与间充质干细胞受到刺激后参与创面修复有关。
AIM： To investigate the effects of serum from burned rats on the gene expression of mesenchymal stem cells in rats cultured in vitro.
METHODS： The experiment was conducted in the Wound Healing and Cell Biology Laboratory, Burn Institute, First Affiliated Hospital of General Hospital of Chinese PLA between June 2003 and April 2004. ①The Wistar rats were killed by decapitation and the femurs were taken to expose the medullary cavity of bones, which was washed with medium, then the mononuelcarecll suspension was extracted with density gradient centrifugation and seeded into flasks to culture mesenehymal stem cells by adherence, ②Rat models of 15% TBSA Ⅲ degree burn was established using 100 ℃ hot water. ③Three days later, blood was collected from abdominal aorta of burned rats to isolate the serum by eentrifugation fraetionation. ④Serum was isolated from normal rats blood as well. ⑤Having been passagcd, mcscnehymal stem cells were then incubated in F-12 medium containing 0.1 volume fraction normal serum （normal serum group） and serum from burn injured rats （burn serum group） for 24 hours and 72 hours, respectively. ⑥Total RNA was extracted from mesenehymal stem cells. The fluorescent eDNA probes with ey3-dUTP and cy5-dUT were prepared through reverse transcription. ⑦The oligonueleotide microarrays containing 5 705 genes were used to detect the differences in gene expression between two groups.
RESULTS：① There were significant differences in different gene expressions between mcsenehymal stem cells of two groups incubated with serum for different time. ②There were 4 genes differentially expressed in two groups after mcscnchymal stem cells incubated for 24 hours. ③There were 11 genes differentially expressed in two groups after mesenchymal stem cells incubated for 72 hours. ④There was no identical gene with differently expressions between the two groups.
CONCLUSION： Serum from burned rats can stimulate mesenchymal stem cells and change the gone expression; moreover, the differently expressed genes change dynamically with the time, which may be related to the wound repair of mesenchymal stem cells after stimulation.
出处
《中国临床康复》
CSCD
北大核心
2006年第37期7-10,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家重点基础研究发展规划资助项目(2005CB522603)
国家自然科学基金(30230370
30400172)~~
