摘要
目的：从JAK2/STAT5信号转导通路探讨黄芪甲苷、毛蕊异黄酮及其配伍对化疗性骨髓抑制小鼠骨髓干细胞的调控机制。方法：C57BL/6小鼠20只,雌雄各半,28~32 g。给模型组小鼠每日腹腔注射一次环磷酰胺380 mg·kg-1,正常小鼠腹腔注射等量生理盐水,连续3 d。造模后,以脱颈椎法处死各组小鼠,取出股骨,剔除肌肉和结缔组织,剪开股骨两端,用6号针头以生理盐水反复冲洗骨髓腔,并将冲出的骨髓打碎,再用4号针头过滤制成单个细胞悬液,在离心机上2000 r/min,离心10 min,去上清液,加入4 m L冷70%乙醇固定,上振荡器摇匀制成骨髓干细胞样本。细胞实验分为6组,包括正常组,模型组,＂EPO＋（G-CSF）＂对照组,毛蕊异黄酮组,黄芪甲苷组,两药配伍组。直接给药黄芪甲苷0.2 mg/m L、毛蕊异黄酮0.01 mg/m L、EPO 50 U/m L、（G-CSF）50 U/m L于骨髓干细胞体外培养体系中,培养3 d,室内18~20℃,20%相对湿度,5%CO2,37℃孵育箱。采用蛋白质印迹Western-blot检测p JAK2和p STAT5的蛋白表达,采用RT-q PCR法检测骨髓干细胞中SOCS3的m RNA表达。结果：（1）对SOCS3m RNA表达的影响。与模型组比较,正常组和各治疗组均有明显差异（P〈0.05）。治疗组之间比较,毛蕊异黄酮组、黄芪甲苷组、两药配伍组3组之间的两两比较均无显著差异（P〉0.05）。与对照组比较,黄芪甲苷组、蕊异黄酮组、两药配伍组均无显著差异（P〉0.05）。（2）对p JAK2蛋白表达的影响。与模型组比较,正常组和各治疗组均有明显差异（P〈0.05）。治疗组之间比较,3组之间的两两比较均无显著差异（P〉0.05）。与对照组比较,毛蕊异黄酮组、两药配伍组均无显著差异（P〉0.05）。（3）对p STAT5蛋白表达的影响。与模型组比较,正常组和各治疗组均有明显差异（P〈0.05）。治疗组之间比较,毛蕊异黄酮组、黄芪甲苷组、两药配伍组3组之间的两两比较均无显著差异（P〉0.05）。与对照组比较,黄芪甲苷组、蕊异黄酮组、两药配伍组均无显著差异（P〉0.05）。结论：黄芪甲苷、毛蕊异黄酮及其配伍通过促进p JAK2、p STAT5蛋白表达,下调SOCS3 m RNA表达,保护化疗后骨髓抑制,促进骨髓造血损伤修复。
Objective： From JAK2 / STAT5 signal transduction pathways to discuss astragalus glycosides and calycosin and the compatibility on bone marrow stem cells due to chemotherapy. Methods： Twenty C57 BL /6 mice,half male and half female,weighted 28 ~ 32 g. The model group mice were given intraperitoneal injection of cyclophosphamide 380 mg /kg once a day and the normal mice given injection amount of normal saline to the abdominal cavity for three consecutive days. After model- building,by cervical vertebra- taking off method we executed the mice. The femur was taken out,eliminating the muscle and connective tissue,opening both ends of the femur. With No. 6 needle,wash the marrow cavity with physiological saline and break the bone marrow. And then with No. 4 needle make the single cell suspension. With the speed of 2000 r / min on centrifuge,centrifuging for 10 minutes to get the clear liquid. Add 4 mL cold 70% ethanol for fixing. By oscillator shake,make samples of bone marrow stem cells. Cells were divided into 6 groups,including normal group,model group,EPO ＋（ G- CSF） control group,calycosin group,astragalus glycosides group,two drugs compatibility group. Directly give astragalus glycosides 0. 2 mg / mL,calycosin separately 0. 01 mg / mL,EPO 50 U / mL and 50 U / mL（ G- CSF） to bone marrow stem cells in vitro culture system for three days,indoor 18 ~ 20 ℃,relative humidity 20%,5%CO2,37℃ incubation box. Using Western blot,detect pJ AK2 and pS TAT5 protein expressions. RT- qP CR method was used to detect SOCS3 mR NA expression in bone marrow stem cells. Results：（ 1） Effects on SOCS3 mR NA expression：Compared with model group,normal group and each treatment group had obvious difference（ P〈0. 05）. Calycosin group,astragalus glycosides group and two drugs compatibility group had no significant difference（ P〉0. 05）. Compared with control group,the astragalus glycosides group,calycosin group and two drugs compatibility group had no significant difference（ P〉0. 05）.（ 2） The effects on pJ AK2 protein expression： Compared with model group,normal group and each treatment groups had obvious difference（ P〈0. 05）. Comparison among treatment groups： The three groups had no significant difference（ P〉0. 05）. Compared with control group,calycosin group and two drugs compatibility group had no significant difference（ P〉0. 05）.（ 3） Effects on pS TAT5 protein expression： Compared with model group,normal group and each treatment group had obvious difference（ P〈0. 05）. Comparison among treatment groups： Calycosin group,astragalus glycosides group and two drugs compatibility groups had no significant difference（ P〉0. 05）. Compared with control group,the astragalus glycosides group,calycosin group and two drugs compatibility group had no significant difference（ P〉0. 05）. Conclusion： Astragalus glycosides,calycosin and its compatibility can protect bone marrow suppression after chemotherapy and promote the bone marrow hematopoietic injury repairing by promoting pJ AK2 and pS TAT5 protein expressions,lowering SOCS3 mR NA expression.
出处
《中华中医药学刊》
CAS
北大核心
2016年第7期1576-1580,共5页
Chinese Archives of Traditional Chinese Medicine
基金
国家自然科学基金项目(81273653)
关键词
黄芪甲苷
毛蕊异黄酮
化疗
骨髓抑制
调控机制
astragalus glycosides
calycosin
chemotherapy
bone marrow suppression
regulatory mechanism
