载阿霉素聚合物微泡影响多发性骨髓瘤癌干细胞增殖凋亡的研究  

DOX-loaded microbubbles inhibit proliferation of multiple myeloma cancer stem cells and induce apoptosis in vitro

作　　者：侍方方[1,2] 李淼[2] 吴松彦 王彩莲[1] 窦骏[2] 

机构地区：[1]东南大学附属中大医院肿瘤科 [2]东南大学医学院病原生物学与免疫学系,南京210009

出　　处：《现代免疫学》2016年第4期305-309,共5页Current Immunology

基　　金：中央高校基本科研业务费专项资金资助(2242015k40016)

摘　　要：制备载阿霉素(DOX)的PLGA微泡(PLGA-MB),观察载药微泡联合超声(US)辐照体外作用于多发性骨髓瘤(MM)癌干细胞(CSC)的效应。采用声振法与乳液法制备载DOX的PLGA微泡,镜下观察其形态,检测其粒径、电位、载药量和包封率。采用免疫磁珠分选法从MM JJN3细胞株分选获得CD138ˉCD34ˉCSC。以不同声强的超声联合空白微泡作用于MM CD138ˉCD34ˉCSC 30s,筛选最佳超声辐照条件。设对照组(I组)、DOX组(II组)、DOX+US组(III组)、DOX-MB组(IV组)、DOX-MB+US组(V组),细胞计数试剂盒(CCK-8)法检测细胞的存活率,流式细胞术检测细胞的凋亡,透射电镜观察细胞超微结构,Western blotting法检测Bcl-2的表达。DOX-MB粒径均一,分散性好,平均粒径461.6±85.6nm,zeta电位-11.4±2.35mv,包封率(56.51±9.82)%,载药率(7.10±1.04)%。1.0 W/cm2的超声作用30s能大量破泡而对细胞增殖无明显影响。各实验组中,V组的细胞存活率[(41.89±1.11)%]较其他组[I组(98.71±1.16)%,IV组(92.56±3.49)%,II组(73.69±3.24)%,III组(71.13±4.03)%]明显降低(P<0.05),V组的细胞凋亡率[(29.05±3.21)%]较其他组[I组(2.33±1.09)%,IV组(3.72±1.58)%,II组(15.14±1.11)%,III组(15.03±3.09)%]明显增强(P<0.05)。V组的Bcl-2蛋白表达(灰度值比率0.23±0.06)较其他各组(I组0.82±0.11,II组0.51±0.08,III组0.58±0.12,IV组0.77±0.05)显著降低(P<0.05)。PLGA微泡是良好的DOX传递载体,联合超声辐照体外作用于多发性骨髓瘤癌干细胞可通过诱导凋亡产生明显的细胞增殖抑制作用。This study aimed to prepare DOX-loaded PLGA microbubbles（DOX-MBs） and investigate the effect of DOX MBs combined with ultrasound on multiple myeloma（MM） cancer stem cells（CSC） in vitro. DOX-MBs prepared by acoustic method and emulsion method were examined under microscope. The size distribution, zeta potential, drug loading efficiency and encap- sulation efficiency were further detected. CD138-CD34-CSC were divided from MM JJN3 cells by immunomagnetic separation. Ultrasound（US） with different intensity was applied to MM CSC in the presence of PLGA MBs for 30s to get the proper ultra- sound parameter. MM CSC were divided into Control group（I）, DOX group（II）, DOX-US group（III）, DOX-MBs group （IV）, DOX-MBs＋US group（V）. The inhibitory effect on cell proliferation was detected by CCK method, and the cell apopto- sis was detected by FCM. The ultrastructure of MM CSC was observed by transmission electron microscope. The expression of Bcl-2 was observed by Western blot. DOX-MBs has good dispersion with the size 461.6 ±85.6nm, zeta potential -11.4± 2. 35mv, encapsulation efficiency（56.51 ± 9.82） %, and drug-loading efficiency （7.10 ± 1.04） %. Ultrasound at 1.0W/cm2 for 30s burst the MBs but did no harm cells. The survival rate was lower in V group[（41. 89±1.11） %] than in other groups[I （98. 71±1.16） %,IV （92.56±3.49） %, II（73.69±3.24） %, III（71.13±4.03） %]（P 〈0. 01）. The apoptosis rate was higher in V Group[（29. 05±3. 21） %] than in other groups[I（2.33±1.09）%, IV（3. 72±1.58）%, II（15.14±1.11）% ,III（15.03± 3.09） %]（P 〈0.01）. Karyopyknosis and karyorrhexis were observed in V Group. The expression of Bcl-2 of V Group was inhibited more obviously than any other groups（P〈0.01） . This study demonstrated that as a drug delivery system, DOX-MBs can inhibit the proliferation and induce the apoptosis of MM CSC when combined with ultrasound.

关 键 词：多发性骨髓瘤 癌干细胞 超声微泡 阿霉素 

分 类 号：R730[医药卫生—肿瘤]