人参皂苷Rg_(1)对移植性白血病模型小鼠的作用

Effect of ginsenoside Rg_(1) on transplanted leukemia mouse model

目的研究人参皂苷Rg_(1)对移植性白血病模型小鼠的作用。方法采用免疫磁性分选法(MACS)从K562细胞中分离、纯化CD34^(+)CD38^(-)人白血病干细胞(CD34^(+)CD38^(-)LSCs),流式细胞术检测分选细胞纯度,台盼蓝染色测定分选细胞活性。将6~8周雌性NOD/SCID小鼠27只随机分为对照组、模型组、人参皂苷Rg_(1)(200 mg/kg)组,每组9只,模型组和人参皂苷Rg_(1)组通过尾iv移植CD34^(+)CD38^(-)LSCs构建白血病小鼠模型,ip给药30 d。观察各组小鼠一般情况及腹部包块变化;全自动血常规检测仪检测外周血白细胞、红细胞、血红蛋白、血小板水平;免疫组化法检测肝、脾脏病理学变化;流式细胞仪分析骨髓细胞周期;CCK-8法检测各组骨髓细胞的增殖能力;细胞免疫荧光染色鉴定骨髓细胞CD34阳性表达情况。结果分选前K562细胞中CD34^(+)CD38^(-)LSCs细胞群百分比为(9.64±1.14)%,分选后CD34^(+)CD38^(-)LSCs纯度可达(96.45±1.63)%;台盼蓝染色显示分选后细胞活性为(95.26±2.16)%。与对照组比较,模型组小鼠腹部包块明显,生存情况较差,体质量显著下降(P<0.05);白细胞数显著增高,红细胞数、血红蛋白水平、血小板数均显著下降(P<0.05);肝、脾脏结构被破坏,有大量白细胞浸润;骨髓细胞周期检测显示G;/G;期比例显著下降(P<0.05),S期比例显著升高(P<0.05);骨髓细胞增殖活力显著升高(P<0.05)。与模型组比较,人参皂苷Rg_(1)组小鼠腹部包块明显减小,生存情况好转,体质量显著增加(P<0.05);白细胞总数显著下降(P<0.05),红细胞数、血红蛋白水平、血小板数均显著升高(P<0.05);肝、脾脏结构明显恢复;骨髓细胞G_(0)/G_(1)期比例显著升高(P<0.05),G_(0)/M期和S期比例显著下降(P<0.05)。细胞免疫荧光检测显示,模型组和人参皂苷Rg_(1)组中小鼠的骨髓细胞中存在大量的人源白血病细胞。结论 NOD/SCID小鼠尾iv移植CD34^(+)CD38^(-)LSCs可成功构建急性髓系白血病小鼠模型,人参皂苷Rg_(1)能有效缓解NOD/SCID小鼠的白血病症状。

Objective To study the effect of ginsenoside Rg_(1)on transplanted leukemia mice model.Methods CD34^(+)CD38^(-)LSCs were isolated and purified from K562 cells by immunomagnetic separation (MACS).The purity of CD34^(+)CD38^(-)LSCs was determined by flow cytometry,and the activity of CD34^(+)CD38^(-)LSCs was determined by Trypan blue staining.Totally 36 female NOD/SCID mice,6—8 weeks old,were randomly divided into control group,model group and ginsenoside Rg_(1)(200 mg/kg) group,with nine mice in each group.The mouse model of leukemia in model group and ginsenoside Rg1 group was established by tail IVtransplantation of CD34^(+)CD38^(-)LSCs,and ip administration of corresponding drugs for 30 days.The general condition and abdominal mass changes of mice in each group were observed.Automatic blood routine detector was used to detect peripheral blood white blood cell,red blood cell,hemoglobin,platelet levels.The pathological changes of liver and spleen were detected by immunohistochemistry.Bone marrow cell cycle was analyzed by flow cytometry.CCK-8 method was used to detect the proliferation of bone marrow cells in each group.CD34 positive expression of bone marrow cells was determined by immunofluorescence staining.Results The percentage of CD34^(+)CD38^(-)LSCs in K562 cells was (9.64±1.14)%before separation,and the purity of CD34^(+)CD38^(-)LSCs reached (96.45±1.63)%after separation.Trypan blue staining showed that the cell activity after separation was(95.26±2.16)%.Compared with control group,abdominal mass was obvious in model group,the survival was poor,and body weight was significantly decreased (P<0.05).WBC count was significantly increased,RBC count,hemoglobin level and platelet count were significantly decreased (P<0.05).The structure of liver and spleen was destroyed,and there were a lot of leukocytes.Bone marrow cell cycle detection showed that the proportion of G;/G;phase was significantly decreased (P<0.05),and that of Sphase was significantly increased (P<0.05).The proliferation activity of bone marrow cells was significantly increased (P<0.05).Compared with model group,abdominal mass in ginsenoside Rg_(1)group was significantly reduced,survival was improved,and body weight was significantly increased (P<0.05).The total number of WBC was significantly decreased (P<0.05),while the RBCnumber,hemoglobin level and platelet count were significantly increased (P<0.05).The structure of liver and spleen recovered significantly.The proportion of G_(0)/G_(1)phase was significantly increased (P<0.05),while the proportion of G_(0)/M and S phase was significantly decreased (P<0.05).Immunofluorescence assay showed that there were a large number of human leukemia cells in bone marrow cells of mice in model group and ginsenoside Rg_(1)group.Conclusion Tail iv transplantation of CD34^(+)CD38^(-)LSCs construct acute myeloid leukemia mouse model,ginsenoside Rg_(1)can effectively alleviate the symptoms of leukemia in NOD/SCID mice.