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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。
(DOX) loaded PLGA microbubbles (PLGA-MB) were prepared and the effects of drug-loaded microbubbles combined with ultrasound (US) irradiation on multiple myeloma (MM) cancer stem cells (CSCs) The DOX-loaded PLGA microbubbles were prepared by sonication and emulsion methods. Morphology was observed microscopically and the particle size, potential, drug loading and entrapment efficiency were measured. CD138ˉCD34ˉCSC was obtained from MM JJN3 cell line by immunomagnetic bead sorting. Ultrasound with different sound intensity combined with blank microbubbles MM CD138 ˉ CD34 ˉ CSC 30s, screening the best ultrasound irradiation conditions. The control group (group I), DOX group (group II), DOX + US group (group III), DOX-MB group (group IV), DOX-MB + -8). Cell apoptosis was detected by flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. The expression of Bcl-2 was detected by Western blotting. The average particle size was 461.6 ± 85.6nm, the zeta potential was -11.4 ± 2.35mv, the entrapment efficiency (56.51 ± 9.82)% and drug loading rate (7.10 ± 1.04)%, respectively. Ultrasound effect of 1.0 W / cm2 for 30s can break a large number of bubbles without any significant effect on cell proliferation. The cell viability in V group was significantly higher than that in other groups [(41.89 ± 1.11)% vs 98.71 ± 1.16% in group I, 92.56 ± 3.49% in group IV, 73.69 ± 3.24% in group II, (71.13 ± 4.03)% in group III (P <0.05). The apoptosis rate in group V [(29.05 ± 3.21)%] was significantly higher than that in other groups 1.58%), Group II (15.14 ± 1.11)%, Group III (15.03 ± 3.09)%]. The expression of Bcl-2 protein in group V was significantly lower than that in other groups (0.82 ± 0.11 in group I, 0.51 ± 0.08 in group II, 0.58 ± 0.12 in group III, and 0.77 ± 0.05 in group IV) (P <0.23 ± 0.06) <0.05). PLGA microbubbles is a good DOX delivery carrier, combined with ultrasound irradiation in vitro on multiple myeloma cancer stem cells can induce apoptosis through cell proliferation inhibition.