复方扶芳藤合剂含药血清对大鼠体外模拟“造血龛”中造血干细胞增殖的影响  

Effects of Compound Fufangteng Mixture Containing Serum on Proliferation of Hematopoietic Stem Cells in Simulated Niche in Vitro of Rats

作　　者：周倍伊 万娟 周围 谢金玲 钟卫干 杨力强 罗眈 黄铠琴 ZHOU Beiyi;WAN Juan;ZHOU Wei;XIE Jinling;ZHONG Weigan;YANG Liqiang;LUO Dan;HUANG Kaiqin(Basic Medical College,Guangxi University of Chinese Medicine,Guangxi Nanning 530200;Faculty of Pharmacy,Guangxi University of Chinese Medicine,Guangxi Nanning 530200;Guangxi Key Laboratory of Efficacy Study on Chinese Materia Medica,Guangxi University of Chinese Medicine,Guangxi Nanning 530200)

机构地区：[1]广西中医药大学基础医学院,广西南宁530200 [2]广西中医药大学药学院,广西南宁530200 [3]广西中医药大学广西中药药效研究重点实验室,广西南宁530200

出　　处：《广西中医药》2020年第1期63-68,共6页Guangxi Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(编号:81460701)。

摘　　要：目的:通过体外共培养大鼠骨髓间充质干细胞(BMSCs)和造血干细胞(HSC)模拟"造血龛",观察BMSCs+HSC Transwell共培养和BMSCs+HSC接触共培养模拟的"造血龛"中BMSCs对HSC生长的影响;将复方扶芳藤合剂含药血清及空白血清加入模拟的"造血龛"中,观察药物对HSC生长的影响。方法:采用全骨髓贴壁法体外培养SD雄性大鼠骨髓间充质干细胞至P3代,流式细胞仪检测其表面标志物,进行BMSCs鉴定。用MACS磁珠分选仪分选SD大鼠骨髓CD90.1+细胞(造血干细胞),流式细胞仪检测其表面标志物,进行HSC鉴定。将BMSCs和HSC共培养:Transwell共培养组(上室接种HSC、下室接种BMSCs),2D接触共培养组(在24孔板同时接种HSC与BMSCs),均连续培养7 d,期间每隔两天使用荧光显微镜观察"龛"中HSC的形态及细胞数量,使用CCK-8法检测"龛"中HSC吸光度值,绘制生长曲线;采用20%浓度的复方扶芳藤合剂含药血清和空白血清连续7 d对各组"龛"中干细胞进行干预,期间每隔两天用使用荧光显微镜观察各组"龛"中HSC的形态及细胞数量,CCK-8法检测各组"龛"中HSC吸光度值,绘制生长曲线,对比各组生长曲线的变化,分析各组"龛"中HSC生长情况。结果:BMSCs具有典型的成纤维样细胞形态,集落生长呈漩涡状。P3代BMSCs表型CD29、CD44、CD34、CD45表达分别为99.709%、99.734%、0.114%、0.013%。HSC在荧光显微镜下激发呈绿色荧光的均一圆形。HSC荧光显微镜下观测及生长曲线显示:各组"龛"中HSC数量均随着培养时间的增加而增加(P<0.05);自第5 d起,Transwell共培养组、2D接触共培养组的HSC吸光度值高于HSC单独培养组(P<0.05);含药血清Transwell共培养组的HSC吸光度值高于空白血清Transwell共培养组(P<0.05),含药血清2D接触共培养组HSC吸光度值高于Transwell共培养组(P<0.05)。结论:全骨髓贴壁法分离培养的BMSCs具有间充质干细胞的生物学特性。MACS磁珠分选可快速获得高纯度HSC;与单独Objective:To observe the effect of Bone marrow Mesenchymal Stem Cells(BMSCs)on proliferation of Hematopoietic Stem Cell(HSC)in the rat simulated niche in vitro in the BMSCs+HSC Transwell coculture mode and the BMSCs+HSC contact coculture mode after co-cultured by rats’BMSCs and HSC.To observe the drug effect on growth of HSC after the compound Fufangteng mixture containing serum and blank serum were applied into the simulated niche in vitro.Methods:The BMSCs in SD rats were cultured to P3 generation by whole bone marrow adherent method.The surface markers of BMSCs were detected by flow cytometry for identification of BMSCs.The bone marrow CD90.1+cells(HSC)of SD rats were selected by Magnetic Cell Sorting(MACS)magnetic sorting instrument.The surface markers were detected by flow cytometry for identification of HSC.The BMSCs and HSCs were co-cultured continuously for 7 days,in the Transwell coculture group the HSC was seeded in the upper chamber while the BMSCs was seeded in the lower chamber.and in the 2D contact coculture group the HSC and BMSCs were seeded in the 24 well plate together.Every 2 days the morphology and cell numbers of HSC were observed by fluorescence microscope,the absorbance value of HSC was detected by CCK-8 method,and the growth curve was recorded.After all the niches were intervened respectively by 20%Compound Fufangteng mixture containing serum and blank serum for 7 days the morphology and numbers of HSC were observed by fluorescence microscope,the absorbance value of HSC was tested by CCK-8 method,the growth curve was drew,and the growth curves of all groups were compared for the analysis of proliferation of HSC.Results:The BMSCs showed typical fibroblast-like cell morphology and dense growth in the form of whirlpool.The expressions of CD29,CD44,CD34 and CD45 were 99.709%,99.734%,0.114%and 0.013%respectively in the P3 generation of BMSCs.The HSC was excited to form a uniform circle with green fluorescence under fluorescence microscope.The number of HSC observed by fluorescence microscope

关 键 词：复方扶芳藤合剂 造血干细胞 骨髓间充质干细胞 造血龛 干细胞增殖 

分 类 号：R285.5[医药卫生—中药学]