当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响

Effect of danggui buxue decoction on the proliferation and c-kit expression of mouse muscle satellite cells in the hematopoietic microenvironment

目的观察当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响。方法分离培养小鼠卫星细胞并鉴定。制备含有不同剂量当归补血汤的大鼠载药血清及对应剂量的骨髓基质细胞条件培养基。将肌卫星细胞随机分为8组:正常大鼠血清组、当归补血汤载药血清1～3组、正常大鼠血清条件培养基组、条件培养基1～3组,MTT法检测细胞增殖活性,免疫组化法检测细胞c-kit的表达情况,荧光实时定量PCR检测细胞c-kit mRNA的表达。结果培养的肌卫星细胞呈Desmin免疫阳性;与正常大鼠血清组相比,载药血清各组及条件培养基各组细胞增殖显著,载药血清3组、条件培养基各组阳性细胞c-kit蛋白及mRNA表达量有显著性差异,随当归补血汤载药血清浓度增大c-kit表达量增多,条件培养基也呈同样的变化趋势。结论当归补血汤载药血清及含载药血清的条件培养基可促进肌卫星细胞增殖及c-kit的表达。

Objective To investigate the effect of danggui buxue decoction on the proliferation and c-kit expression of mouse muscle satellite cells（MSCs） in hematopoietic environment in vitro.Methods MSCs from new born mice were cultured and identified.Serum contained of danggui buxue decoction and the marrow stroma cell conditioned medium were prepared.MSCs were divided into 8 groups,and then MSCs were cultured in the different concentration serum containing drug of danggui buxue decoction and in the marrow stroma cell conditioned medium disposed by the different concentration serum containing drug.MTT method was used to observe the proliferation of MSCs.The c-kit protein of MSCs was examined by immunohistochemistry.The c-kit mRNA of MSCs was detected by real time fluorescence quantitative RT-PCR method.Results The desmin protein was positive in the cultured MSC.After the stimulation of different culture fluid,MSCs of danggui buxue decoction containing serum groups and marrow stroma cell conditioned medium groups were proliferated more than those of normal rat serum control group.MSCs of danggui buxue decoction containing serum group 3 and the marrow stroma cell conditioned medium groups expressed more c-kit than those of normal rat serum control group.Danggui buxue decoction containing serum promoted c-kit expression of the MSCs in a dose-dependent manner,and MSCs of conditioned medium groups were the same trend with the danggui buxue decoction containing serum groups.Conclusions Danggui buxue decoction containing serum and the marrow stroma cell conditioned medium disposed by the serum containing drug could promote MSCs to proliferate and express c-kit gene.