当归多糖对人白血病干细胞体外增殖与体内移植模型的影响  

Effects of Angelica Sinensis polysaccharide on proliferation in vitro and transplantation of human leukemia stem cells in vivo

作　　者：邓方芳 耿珊[1] 姜蓉[1] 汪子铃 肖含先之 齐嵘嘉 黄彩虹 曾娣[1] 李赓 王璐[1] 王亚平[1] DENG Fang-fang;GENG Shan;JIANG Rong;WANG Zi-ling;XIAO Han-xian-zhi;QI Rong-jia;HUANG Cai-hong;ZENG Di;LI Geng;WANG Lu;WANG Ya-ping(Laboratory of Stem Cell and Tissue Engineering,Department of Histology and Embryology,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学干细胞与组织工程研究室,组织学与胚胎学教研室,重庆400016

出　　处：《解剖学报》2021年第1期41-48,共8页Acta Anatomica Sinica

基　　金：国家自然科学基金(81673748,81873103)。

摘　　要：目的探讨当归多糖(ASP)对人白血病干细胞(LSCs)增殖及体内移植的影响。方法1.ASP对CD34^(+)CD38^(-)人LSCs体外增殖的影响。免疫磁性法分选正常人和髓系白血病患者骨髓中CD34^(+)CD38^(-)细胞,分为正常CD34^(+)CD38^(-)对照组、CD34^(+)CD38^(-)LSCs对照组、正常CD34^(+)CD38^(-)ASP组和CD34^(+)CD38^(-)LSCs ASP组,后两组在常规培养基础上加入ASP(终浓度40 mg/L)。流式细胞术检测CD34^(+)CD38^(-)细胞纯度;锥虫蓝染色检测细胞存活率;细胞计数试剂盒8(CCK-8)和混合集落形成单位(CFU-Mix)检测CD34^(+)CD38^(-)LSCs增殖分化能力;RT-PCR检测衰老相关基因表达水平。2.ASP对人LSCs移植小鼠模型的影响。体内移植LSCs建立CD34^(+)CD38^(-)LSCs小鼠移植模型,随机分为ASP移植模型组[腹腔注射ASP,200 mg/kg,(1次/d)×14 d]和移植模型组(注射等时、等量生理盐水)。药物注射完成第2天,计数白细胞总数和分类计数,观察血细胞形态;免疫荧光细胞化学法检测受体鼠骨髓中CD34^(+)CD38^(-)LSCs;衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测染色阳性骨髓单核细胞(BMMCs)百分率;流式细胞术检测BMMCs细胞周期分布;CFU-Mix培养检测BMMCs集落形成能力。结果分选后CD34^(+)CD38^(-)细胞纯度达到(91.14±1.02)%,细胞存活率为(95.42±3.5)%;CD34^(+)CD38^(-)LSCs对照组细胞增殖与集落形成能力显著高于正常CD34^(+)CD38^(-)对照组;CD34^(+)CD38^(-)LSCs ASP组细胞增殖能力与集落形成能力明显低于CD34^(+)CD38^(-)LSCs对照组;CD34^(+)CD38^(-)LSCs ASP组细胞高表达p16^(INK4a)、p19^(Arf)、p53、p21^(Cip1/Waf1)基因。移植CD34^(+)CD38^(-)LSCs后小鼠骨髓中存在人源性LSCs。移植模型组小鼠白细胞总数增加,中性粒细胞比例升高,淋巴细胞比例降低;注射ASP可明显降低白细胞总数和中性粒细胞比例,淋巴细胞比例有所升高;处于G0/G1期中的BMMCs数量增加,S期细胞数量减少;SA-β-Gal染色阳性细胞百分率明显提高;CFU-Mix集落形�Objective To investigate the effect of Angelica Sinensis polysaccharide(ASP)on proliferation,differentiation and transplantation of human leukemia stem cells(LSCs).Methods 1.Effect of angelica sinensis polysaccharides on proliferation of CD34^(+)CD38^(-)human LSCs in vitro.By using immunomagnetic bead sorting,CD34^(+)CD38^(-)cells isolated from the bone marrow of normal persons and myeloid leukemia patients were divided into normal CD34^(+)CD38^(-)group,CD34^(+)CD38^(-)LSCs group,CD34^(+)CD38^(-)ASP group and CD34^(+)CD38^(-)LSCs ASP group.The latter two groups were added ASP(terminal concentration 40 mg/L)on the basis of conventional culture.The purity of CD34^(+)CD38^(-)cells was detected by flow cytometry.The viability of cells was detected by trypan blue staining.The proliferation of CD34^(+)CD38^(-)cells were detected by cell counting kit 8(CCK-8)and colony forming unit-mixture(CFU-Mix).The expression of agingrelated gene was detected by RT-PCR.2.Effect of Angelica Sinensis polysaccharide on mouse model of LSCs transplantation.After to establish CD34^(+)CD38^(-)LSCs transplantation mice model,the mice were randomly divided into ASP transplantation model group(intraperitoneal injection with ASP,200 mg/kg,Qd×14 days)and transplantation model group(intraperitoneal injection with the same volume of saline at the same time).On the 2nd day after drug injection was finished,leukocytes count and classification and cells morphology were observed.By using immunofluorescence cytochemistry,CD34^(+)CD38^(-)LSCs in bone marrow of recipient mice was detected.The percentage of senescence assosiatedβ-galactosidase(SA-β-Gal)positive staining cells in bone marrow mononuclear cells(BMMCs)was detected.Cell cycle distribution of BMMCs was detected by flow cytometry.The colony formation ability of BMMCs was detected by CFU-Mix.Results The purity of CD34^(+)CD38^(-)cells was(91.14±1.02)%and the viability of cells was(95.42±3.52)%.The proliferation and CFU-Mix formation ability of CD34^(+)CD38^(-)LSCs group were significantly h

关 键 词：当归多糖 人白血病干细胞 增殖抑制 体内移植 细胞衰老 流式细胞术 小鼠 

分 类 号：R285[医药卫生—中药学]