睾酮诱导的小鼠骨髓巨噬细胞凋亡途径研究

用睾酮诱导,研究小鼠骨髓巨噬细胞(BMMs)凋亡过程中Fas/FasL途径上caspase-8表达的改变。以BMMs经L929条件培养基(LCM)诱导5d后,用流式细胞仪分选出F4/80阳性细胞,并将细胞分为两大组。第1组是空白对照组,睾酮(100nmol·L-1,下同)处理组,去除LCM组,去除LCM同时用睾酮处理组。第2组用FADD反义寡核苷酸(ASODN)转染BMMs后,重复第1组的4个处理组,并以FADD错义寡核苷酸(MSODN)转染后的睾酮处理组作为对照。处理12h后,用流式细胞仪检测巨噬细胞的凋亡情况,并通过RT-PCR、Real-timeRT-PCR和WesternBlot方法检测各组中caspase-8基因及蛋白的表达。结果显示,在缺少LCM或用睾酮处理时,睾酮可在体外诱导小鼠骨髓巨噬细胞凋亡,并伴随caspase-8的活化。FADD反义寡核苷酸能抑制睾酮诱导的小鼠骨髓巨噬细胞的凋亡,其下游的caspase-8表达也被抑制。

Investigation of the Pathway of Apoptosis Induced by Testosterone in Bone Marrow-derived Macrophages

To delineate the effect of testosterone on the apoptosis of bone marrow-derived macrophages(BMMs)and the variant expression of caspase-8 during Fas/FasL pathway.BMMs were cultured with LCM for 5 days to differentiate towards macrophages.F4/80 positive cells were sorted by flow cytometry and divided into two groups.In one group,BMMs were stimulated with testosterone at the presence of LCM or not.In another group,BMMs were transfected with ASODN to FADD and treated similar to the first group.FADD-MSODN treatment was acted as a control.After 12h,Flow Cytometry was used to quantify the apoptosis of BMMs.Real-time RT-PCR and Western Blot were performed to analyze the expression of caspase-8 during the Fas/FasL pathway.The data showed that testosterone could induce the apoptosis of BMMs,similar to removing growth factor M-CSF from the culture medium.They were both associated with the enhanced expression of caspase-8.The blockage of FADD could decrease the expression of caspase-8 obviously in apoptotic macrophages induced by testosterone.These results suggest that Fas/FasL pathway may play an important role in the testosterone-induced apoptosis of macrophages.