Objective: To explore the difference in the proliferation inhibition of doxorubicin and dual specific oncolytic adenoviruses (Ad-VT, Ad-T, Ad-VP3 and d-Mock) on breast cancer cells and normal mammary cells. Methods: The proliferation inhibition rates of doxorubicin and recombinant adenovirus(Ad-VT, Ad-T, Ad-VP3and Mock) on breast cancer cells were detected through WST-1 experiment, and the effects of two drugs on the inhibitory rates of normal mammary epithelial cells were also detected. Moreover, the apoptosis rates of doxorubicin and oncolytic adenoviruses on breast cancer cells and normal mammary epithelial cells were evaluated by Annexin V flow cytometry, Hoechst and JC-1 staining, and the difference in the apoptosis rates were also compared. Results: All the recombinant adenovirus could effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), the inhibition effects followed the order ofAd-VT>Ad-T>Ad-VP3>Ad-MOCK, and the inhibition effect was positively correlated with time. Doxorubicin could also effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), and the inhibition effect was markedly enhanced with the increases in does and time. However, doxorubicin also showed strong inhibition effect on the normal mammary epithelial cells, and the inhibition rate achieved 80% under 72 h and 5 ug/ml doxorubicin, while that of oncolytic adenovirus Ad-VT on MCF-10A was 20% at 72 h. The apoptosis effects of oncolytic adenoviruses-induced breast cancer cellwere increased with time, and the apoptosis rate efficiency followed the order of Ad-VT>Ad-T>Ad-VP3>Ad-MOCK, but they displayed low ability to induce normal mammary cell apoptosis. The apoptosis effects of doxorubicin-induced breast cancer cell were similar to that of the normal mammary epithelial cell (P <0.05 or P<0.01), which followed the dose of 0.05<0.5<5 μg/ml. Conclusion: Dual specific oncolytic adenoviruses can effectively suppress the proliferation of breast cancer cells, but they have low inhibition on normal mammary cells, which have displayed superior safety and provide a new method for the biotherapy of tumor.

[摘 要] 目的：设计并制备一种分别靶向B细胞表面抗原CD19和CD22的CAR-T细胞，检测其对肿瘤细胞的体内外杀伤效果。方法：将含有人源化 CD19 ScFv的二代CAR分子和带有CD3ε链作为共刺激结构域的CD22 ScFv CAR分子以P2A自剪切肽连接，序列连接于慢病毒载体pLTR-CMV-MCS中，以HEK-293T细胞包装相应的慢病毒载体，感染健康志愿者提供的T细胞制备CAR-19-22-T细胞，同时以相同二代结构分别构建单靶向CAR-T细胞作为参照。构建表达荧光素酶、CD19和/或CD22的前列腺癌3M细胞（靶细胞）。将各种CAR-T细胞与靶细胞共同培养，采用荧光素酶化学发光法和ELISA法检测其对靶细胞的杀伤能力和细胞因子的分泌水平。通过尾静脉注射Raji-Luc细胞构建NOD-SCID免疫缺陷小鼠白血病模型，分别注射各组CAR-T细胞进行治疗并评估其疗效。结果：培养7 d的CAR-19-22-T细胞的CAR-19表达率为13.7%，CAR-22表达率为14.3%。CAR-19-22-T细胞在10∶1效靶比时，对3M-CD19-Luc、3M-CD22-Luc和3M-CD19-CD22-Luc细胞的杀伤率均显著高于T细胞[（78.1±14.4）% vs （11.1±4.3）%、（46.7±10.7）% vs （12.4±2.7）%、（90.5±4.3）% vs （14.3±3.7）%，均P<0.01]；与3M-CD19-Luc、3M-CD22-Luc、3M-CD19-CD22-Luc靶细胞共培养后，CAR-19-22-T细胞IFN-γ、TNF-α和IL-2水平均显著低于CAR-19-T和CAR-22-T细胞（P<0.05或P<0.01）。CAR-19-22-T细胞对移植Raji-Luc细胞模型小鼠治疗效果明显，其生存期显著长于T细胞组（P<0.01），与CAR-19-T组和CAR-22-T组荷瘤小鼠比较差异均无统计学意义（均P>0.05）。结论：成功设计并制备了一种双靶点CAR-19-22-T细胞，其能够有效杀伤表达CD19和/或CD22抗原的肿瘤细胞，对Raji-Luc细胞的白血病模型小鼠有显著的治疗效果。

[摘 要] 目的：探讨光甘草定对肺腺癌细胞A549恶性生物学行的影响及其分子机制。方法：常规方法培养A549细胞和人正常肺上皮细胞BEAS-2B，用不同浓度的光甘草定和或顺铂对其进行处理，通过结晶紫染色、CCK-8法检测光甘草定、顺铂对A549、BEAS-2B细胞增殖活力的影响，Transwell小室实验、细胞划痕实验检测光甘草定对A549细胞侵袭、迁移能力的影响，流式细胞术检测光甘草定对A549细胞凋亡的影响，3D超低黏附板培养法培养A549细胞后采用CCK-8法检测光甘草定对A549细胞增殖的影响，WB法检测光甘草定对A549细胞中上皮间质转化（EMT）相关蛋白表达的影响；构建A549细胞移植瘤模型后检测光甘草定、顺铂对移植瘤的生长以及移植瘤组织中EMT相关蛋白表达的影响。结果：光甘草定、顺铂呈剂量依赖性地显著抑制A549细胞的增殖（P<0.05或P<0.01）、细胞迁移（P<0.05或P<0.01）和侵袭能力（P<0.05或P<0.01），光甘草定能诱导A549细胞凋亡（P<0.01），抑制A549细胞中N-cadherin、snail和vimentin蛋白的表达，促进E-cadherin蛋白表达；光甘草定、顺铂均能抑制A549移植瘤的生长，抑制移植瘤组织中Ki67、N-cadherin、snail和vimentin蛋白的表达、促进E-cadherin蛋白的表达。结论：光甘草定可通过抑制A549细胞的增殖、迁移和侵袭，诱导A549细胞凋亡，其机制可能与其抑制细胞的EMT进程而产生抑癌作用有关。