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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: To investigate the regulatory mechanism of astragaloside, calycosin and their compatibility on bone marrow stem cells in mice with chemotherapy-induced myelosuppression from the JAK2 / STAT5 signal transduction pathway. Methods: Twenty C57BL / 6 mice were divided into two groups, male and female, 28 ~ 32 g. The model mice were intraperitoneally injected with cyclophosphamide (380 mg · kg -1) once a day, and the normal mice were injected with the same amount of normal saline intraperitoneally for 3 days. After the model was established, all the mice were sacrificed by cervical dislocation, the femurs were removed, the muscles and connective tissue were removed, both ends of the femur were cut off, the bone marrow cavity was repeatedly washed with physiological saline with a 6-gauge needle, Then filtered through a 4-pin needle to make a single cell suspension, centrifuged at 2000 r / min for 10 min in a centrifuge, the supernatant was removed, 4 mL of cold 70% ethanol was added, and shaker was used to sharpen bone marrow stem cell samples . Cell experiments were divided into 6 groups, including normal group, model group, “EPO + (G-CSF)” control group, calycosin group, astragaloside group, Direct administration of astragaloside 0.2 mg / m L, calycosin 0.01 mg / m L, EPO 50 U / m L, and (G-CSF) 50 U / m L were cultured in vitro for 3 days, 18 ~ 20 ℃, 20% relative humidity, 5% CO2, 37 ℃ incubator. The protein expression of p JAK2 and p STAT5 was detected by Western blotting. The mRNA expression of SOCS3 in bone marrow stem cells was detected by RT-q PCR. Results: (1) Effect on SOCS3m RNA expression. Compared with the model group, the normal group and each treatment group were significantly different (P <0.05). There was no significant difference (P> 0.05) between the two groups in the three groups of calycosin, astragaloside group and two drug compatibility groups. Compared with the control group, Astragaloside group, core isoflavone group, two drug compatibility group had no significant difference (P> 0.05). (2) Effect on p JAK2 protein expression. Compared with the model group, the normal group and each treatment group were significantly different (P <0.05). There was no significant difference between the two groups (P> 0.05). Compared with the control group, there was no significant difference between the calycosin group and the two drugs compatibility group (P> 0.05). (3) Effect on p STAT5 protein expression. Compared with the model group, the normal group and each treatment group were significantly different (P <0.05). There was no significant difference (P> 0.05) between the two groups in the three groups of calycosin, astragaloside group and two drug compatibility groups. Compared with the control group, Astragaloside group, core isoflavone group, two drug compatibility group had no significant difference (P> 0.05). CONCLUSIONS: Astragaloside, calycosin and their combination can promote the bone marrow hematopoietic injury by promoting the expression of p JAK2 and p STAT5, down-regulating the expression of SOCS3 m RNA, protecting myelosuppression after chemotherapy.