当归多糖促进骨髓移植小鼠造血功能重建及其机制研究

Effect of Angelica sinensis polysaccharides on hematopoietic reconstitution in mice after bone marrow transplantation and its mechanism

目的探讨当归多糖(Angelica sinensis polysaccharides,ASP)对接受供体骨髓细胞移植后受体小鼠的造血功能重建及其初步机制。方法取10只20~25 g雄性C57BL/6J小鼠作为供体小鼠,提取其骨髓细胞;将30只8~10周龄同系受体雌性小鼠经8.0 Gy的X射线(一次性致死剂量)全身辐射后,将供体小鼠的骨髓细胞通过尾静脉输入受体小鼠体内来构建小鼠骨髓移植模型,随后采用简单随机化法将受体小鼠分为3组,每组10只。对照组(Ctrl组):受体小鼠辐射后不移植骨髓细胞;骨髓移植组(BMT组):受体小鼠辐射后移植骨髓细胞(5×10^(6)个/只);骨髓移植+当归多糖组(TASP组):辐射剂量与移植骨髓细胞数同骨髓移植组,同时给受体小鼠腹腔注ASP(100 mg/kg×9 d)。期间动态测量受体小鼠体质量变化。移植骨髓细胞后第9天,PCR方法鉴定受体鼠脾集落细胞和骨髓细胞的Y染色体基因。采集受体小鼠眼眶血,检测白细胞(WBC)、红细胞(RBC)、血红蛋白(HGB)、血小板(PLT)数量。取受体小鼠股骨,计数每只股骨有核细胞数,流式细胞术检测骨髓细胞周期。培养受体鼠骨髓基质细胞,观察基质细胞贴壁情况,EDU法检测骨髓基质细胞增殖能力,并计数成纤维细胞集落生成单位(CFU-F)。ELISA法检测受体鼠血清和骨髓基质细胞培养上清液中相关造血因子含量。结果对照组小鼠7 d内全部死亡,BMT组与TASP组小鼠全部存活。两个移植组的小鼠体质量5~6 d降至最低后开始恢复,TASP组恢复更快(P<0.05)。在雌性受体小鼠脾集落细胞和骨髓造血细胞中检测到Y染色体基因。TASP组小鼠外周血WBC、RBC、HGB数量与每只股骨有核细胞数均显著高于BMT组(P<0.05;P<0.01),且骨髓细胞G_(0)/G_(1)期细胞比例下降,S期和G_(2)/M期细胞比例增高(P<0.05)。TASP组受体小鼠的骨髓基质细胞贴壁数量、增殖能力和形成CFU-F集落数量也明显高于BMT组(P<0.05),且小鼠血清粒-巨噬细胞集落刺激因子(GM-CSF)和白介素3(IL-3)以及骨髓基质细胞培养上清液中GM-CSF和干细胞因子(SCF)含量显著增高(P<0.05)。结论ASP可促进移植骨髓细胞在受体小鼠体内造血功能的重建,其初步机制可能与改善造血微环境和促进造血因子分泌有关。

Objective To investigate the effect and mechanism of Angelica sinensis polysaccharides(ASP)on the hematopoietic reconstitution of recipient mice after bone marrow transplantation.Methods Bone marrow cells were derived from 10 male C57BL/6J mice(20~25 g),and 30 female recipient mice(8~10 weeks old)were irradiated with 8.0 Gy lethal dose of X-ray.The bone marrow cells from donor mice were infused through tail vein to construct the mouse bone marrow transplantation model.The recipient mice were randomly divided into 3 groups with 10 mice in each group:control group(Ctrl):no bone marrow cells were transplanted in recipient mice;Bone marrow transplantation group(BMT):recipient mice were transplanted with bone marrow cells of donor mice(5×10^(6)/each);Bone marrow transplantation+Angelica sinensis polysaccharide group(TASP):radiation dose and the numbers of transplanted bone marrow cells were the same as those of BMT group.At the same time,the recipient mice were given the ASP by intraperitoneal injection(100 mg/kg×9 d).The change in body weight of recipient mice was measured dynamically.In the ninth day after bone marrow cells transplantation,Y chromosome genes in colony-forming cells in spleen and bone marrow cells of recipient mice were detected by PCR.Blood samples were collected from the orbit of the recipient mice to measure the number of WBC,RBC and PLT and the level of HGB.Femurs were taken after the recipient mice were killed,and the number of bone marrow nucleated cells(BMNC)in each femur was counted.The bone marrow cell cycle was detected by flow cytometry.Bone marrow stromal cells(BMSCs)were cultured in vitro.The adherent status of BMSCs was observed,cell proliferative capacity was detected by EDU,and colony forming unit-fibroblastic(CFU-F)numbers were counted.The contents of hematopoietic factors in serum and supernatant of BMSCs were determined by ELISA.Results All mice in the control group died within 7 d,and all animals in the other groups survived.The body weight of mice in the BMT and TASP groups decreased to the lowest level and began to recover after 5~6 d,and the TASP group showed a faster recovery in body weight(P<0.05).Y chromosome genes were detected in colony-forming cells in spleen and bone marrow cells of recipient mice.The number of WBC,RBC and the level of HGB in the TASP group were increased compared with the BMT group(P<0.01;P<0.05).The number of BMNCs in each femur in the TASP group were significantly higher than those in the BMT group(P<0.05).In the TASP group,the cell proportion decreased in the G_(0)/G_(1)phase,and the cell proportion in the S and G_(2)/M phase increased compared with the BMT group(P<0.05);The adherent numbers of BMSCs,proliferation ability and CFU-F numbers in the TASP mice were also significantly higher than those in the BMT group(P<0.05).The contents of granulocyte-macrophage colony stimulating factor(GM-CSF)and interleukin-3(IL-3)in serum and GM-CSF and stem cell factor(SCF)in supernatant of BMSCs were increased in the TASP group compared with the BMT group(P<0.05).Conclusion ASP can promote hematopoietic reconstitution in recipient mice after bone marrow cells transplantation of donor mice,and the preliminary mechanism may be related to improving hematopoietic microenvironment and promoting secretion of hematopoietic factors.