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目的探讨以重组质粒pc DNA3.1+-CD44(s1-s5)转染的树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)联合培养后对乳腺癌干细胞(BCSC)的体外杀伤效应。方法传代培养乳腺癌细胞株MDA-MB-231,构建重组质粒p ET32aCD44(s1-s5)及pc DNA3.1+-CD44(s1-s5),前者转化感受态BL21后经原核系统表达,检测蛋白相对分子质量;后者通过化学转染法负载正常人外周血单个核细胞分离诱导的DC后,与同源诱导成熟的CIK细胞共同培养,以此为实验组[CD44(s1-s5)-DC-CIK组],并与对照组(载体质粒-DC-CIK组),即同样条件下以载体质粒pc DNA3.1+转染的DC与CIK共同培养,对体外微球体培养法富集的乳腺癌干细胞进行杀伤,通过乳酸脱氢酶(LDH)释放法检测各组的LDH释放值,计算细胞毒性百分比来对比两组效应细胞对靶细胞的杀伤效果。结果重组质粒构建成功,并能通过原核及真核系统正常表达;经过6 d的诱导培养,DC表面伸出大量突起,形态趋于成熟,CIK细胞始终呈单一圆球形悬浮生长,细胞数目较前明显增多。用LDH释放法检测在40∶1、20∶1、10∶1、5∶1 4种效靶比,在相同效靶比时,CD44(s1-s5)-DC-CIK组细胞杀伤活性(分别为33.45%±1.24%、25.43%±0.51%、16.07%±1.16%和9.40%±1.08%)明显高于载体质粒-DC-CIK组(分别为17.56%±0.71%、12.97%±0.36%、7.62%±0.25%和5.13%±0.21%),差异均有统计学意义(P<0.05)。结论乳腺癌干细胞抗原负载的DC可以提高CIK细胞对抗原相关乳腺癌细胞的靶向杀伤活性,为临床生物靶向治疗的开展奠定了实验基础。
OBJECTIVE: To investigate the in vitro killing of breast cancer stem cells (BCSC) after co-culture of dendritic cells (DCs) transfected with recombinant plasmid pcDNA3.1 + -CD44 (s1-s5) and cytokine-induced killer cells effect. Methods The recombinant plasmid p ET32aCD44 (s1-s5) and pcDNA3.1 + -CD44 (s1-s5) were sub-cultured and subcultured. The former was transformed into competent cells BL21 and expressed in prokaryotic cells. Relative molecular mass. The latter was transfected with normal human peripheral blood mononuclear cells (DCs) by chemical transfection method, and then co-cultured with homozygous CIK cells. As a result, CD44 (s1-s5) -DC -CIK group], and compared with the control group (vector plasmid-DC-CIK group), that is, DCs transfected with the pcDNA3.1 + vector under the same conditions were co-cultured with CIK, and the mammary glands enriched by in vitro microsphere culture Cancer stem cells were killed. The release of LDH in each group was measured by lactate dehydrogenase (LDH) release method. The cytotoxicity percentage was calculated to compare the killing effect of two groups of effector cells on target cells. Results The recombinant plasmids were successfully constructed and expressed normally in prokaryotic and eukaryotic systems. After 6 days of induction, numerous protuberances appeared on the surface of DCs and the morphology tended to mature. CIK cells were always grown in a single round spherical suspension with a higher number of cells Significantly increased. The cytotoxicity of CD44 (s1-s5) -DC-CIK group at 40: 1, 20: 1, 10: 1, 5: 1 and 4: (33.45% ± 1.24%, 25.43% ± 0.51%, 16.07% ± 1.16% and 9.40% ± 1.08%) were significantly higher than that of vector plasmid-DC-CIK group (17.56% ± 0.71%, 12.97% ± 0.36% 7.62% ± 0.25% and 5.13% ± 0.21% respectively), the difference was statistically significant (P <0.05). Conclusion DCs loaded with breast cancer stem cell antigen can enhance the cytotoxicity of CIK cells against antigen-related breast cancer cells and lay the foundation for the development of clinical bio-targeted therapy.