摘要
目的:研究负载α-半乳糖神经酰胺(alpha-galactosylceramide,α-Gal Cer)的DC功能与成熟度的改变情况,探讨α-Gal Cer-DC与CIK共培养对CIK细胞表型、增殖活性及杀伤肝癌细胞效率的影响。方法:采用密度梯度离心法从人外周血中分离出单个核细胞,悬浮细胞诱导培养CIK细胞,贴壁细胞诱导培养DC;流式细胞仪检测α-Gal Cer负载DC的表型,Real-time PCR检测DC相关基因的mRNA表达改变。将α-Gal Cer-DC与CIK细胞共培养,流式细胞仪检测DC与CIK细胞表面标志物;锥虫蓝染色法检测CIK细胞的增殖倍数;Real-time PCR检测CIK细胞功能相关基因的表达情况;CCK-8试剂盒检测α-Gal Cer-DC对CIK杀伤Hep G2细胞的影响。结果:经过多种细胞因子诱导,可获得CIK细胞和成熟的DC;α-Gal Cer负载可促进DC成熟,DC表面标志物CD80、CD86、CD83和CD11c的阳性率均升高(P<0.05或P<0.01),表面趋化因子受体CCR-7、IL-12、IL-10的mRNA水平升高(P<0.05)。α-Gal Cer-DC与CIK共培养,可显著提高CIK细胞CD3+CD56+的表达和增殖活性(P<0.05或P<0.01),并显著提高INF-γ、IL-12、穿孔素和颗粒酶素B的mRNA表达水平(P<0.05或P<0.01);CIK、DC-CIK、α-Gal Cer-DC-CIK细胞对Hep G2细胞的杀伤作用随效靶比的升高而增强,在同一效靶比时α-Gal Cer-DC-CIK细胞对靶细胞的杀伤作用最强(P<0.05)。结论:α-Gal Cer负载可促进DC成熟,α-Gal Cer-DC与CIK共培养能促进后者增殖和成熟,能显著增强其对肝癌细胞的杀伤活性,为DC-CIK在肿瘤免疫治疗中的应用提供了实验依据。
Objective： To investigate function and maturation of DCs pulsed by α-Gal Cer and to explore influence of co-culture of CIK cells with α-Gal Cer-DCs on phenotype,proliferation activity and efficiency of killing hepatic carcinoma of the CIK cells. Methods： DCs and CIK cells were induced from monocytes that separated from human peripheral blood by density gradient centrifugation. The suspension and adherent monocytes were cultured ex vivo to generate DC and CIK cells respectively. The phenotypes of DCs pulsed by α-Gal Cer were detected by flow cytometry（ FCM）,and mRNA expression of related genes in DCs were detected by qRT-PCR. As co-culture of CIK cells with α-Gal Cer-DCs,surface markers on the DC and the CIK cells were detected by FCM. Proliferation multiple of the CIK cells was examined with trypan blue staining; expression levels of genes related with function of the CIK cells were measured by Real-time PCR;and effect of α-Gal Cer-DCs on killing Hep G2 cells by the CIK cells was tested with CCK-8 kit. Results： CIK cells and mature DCs could be obtained from induction of multiple cytokines; Excitation of α-Gal Cer could promote DCs maturation,increase the positive expressions of CD80,CD86,CD83 and CD11c（ P〈0. 05,P〈0. 01） and mRNA levels of surface chemokine receptors CCR-7,IL-12,and IL-10 in the DCs（ P〈0. 05）. Co-cuture of the CIK cells with α-Gal Cer could significantly increase expression of CD3＋CD56＋and proliferation activity of the CIK cells（ P〈0. 05,P〈0. 01）,and mRNA expression levels of INF-γ,IL-12,perforin and particle enzyme B（ P〈0. 05,P〈0. 01）; Killing effects of the CIK,the DC-CIK and the α-Gal Cer-DC-CIK cells on Hep G2 cells were enhanced with increase of effector-target ratio. At the same effector-target ratio,α-Gal Cer-DC-CIK cells had the highest cytotoxicity effect on Hep G2 cells（ P〈0. 05）.Conclusion： Pulse of α-Gal Cer could promote the maturation of DCs. Co-culture of the α-Gal Cer-DC with CIK cells could enhance proliferation and
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2016年第2期175-181,共7页
Chinese Journal of Cancer Biotherapy
基金
山东省科技发展计划资助项目(No.2012GGA01219)
济南市科技发展计划资助项目(No.201302032)
济南市2012年度科技型中小企业技术创新基金资助~~
关键词
树突状细胞
细胞因子诱导的杀伤细胞
Α-GAL
CER
肝癌
增殖
成熟
肿瘤杀伤活性
dendritic cell（DC）
cytokine induced killer（CIK） cell
α-galactosylceramide（α-Gal Cer）
hepatoma carcinoma
proliferation
maturation
tumor cytotoxic effect
