摘要
目的探讨在细胞因子诱导的杀伤细胞(CIK)表达特异性抗原的肿瘤细胞过程中,是否存在抗原特异性杀伤。方法分离健康人骨髓获得单个核细胞,分别诱导为树突状细胞(DC)和CIK细胞,将人类乳腺癌耐药细胞株MCF-7/ADR细胞的冻融物抗原冲击或未冲击DC与CIK细胞共培养(pulsed-DC+CIK、DC+CIK),CIK细胞单独培养作对照。用流式细胞仪分析细胞表型,用酶联免疫吸附法(ELISA)检测IL-12、和IFN-γ分泌水平,用二苯基溴化四氮唑蓝(MTT)法测定细胞毒效应。结果DC与CIK共育后,两组DC成熟表型较共育前明显提高(P=0.003、P=0.001);pulsed- DC+CIK组与DC+CIK组、CIK组比较,细胞表型(CD3、CD8、CD56)明显提高(P=0.003、P= 0.011),CD3+CD56+细胞明显增多(P=0.001,P<0.001),CD3+CD8+细胞亦明显增多(P=0.002, P=0.002);CD45RA表型则明显降低(P<0.001,P=0.004)。IL-12和IFN-γ水平在pulsed-DC+ CIK组表达最高,分别为(254±14.5)pg/ml和(3100±286)pg/ml。对有耐药抗原表达的MCF-7/ADR细胞,pulsed-DC+CIK组杀伤效应最强,pulsed-DC+CIK组、DC+CIK组和CIK组比较,差异均有统计学意义(pulsed-DC+CIK组与DC+CIK组比较,P=0.039;pulsed-DC+CIK组与CIK组比较,P= 0.002;DC+CIK组与CIK组比较,P=0.049);而对于无P-gp抗原表达的MCF-7细胞的杀伤效应,pulsed- DC+CIK组和DC+CIK组之间无明显差异,但均高于CIK组,差异有统计学意义(pulsed-DC+CIK组与CIK组比较,P=0.007;DC+CIK组与CIK组比较,P=0.048)。结论从人骨髓培养得到DC和CIK细胞共培养后,能促进各自特征性表面标志的表达上调,并分泌大量相关细胞因子。细胞杀伤效应的明显提高及可能的特异性细胞杀伤效应,为多药耐药肿瘤的临床生物免疫治疗提供实验基础。
Objective A lot of studies have suggested that a certain amount of T cells may be involved among cytokine-induced killer （CIK） cells. The aim of the present study was to prove whether an antigen-specific killing effect on tumor cells is involved during the CIKs-induced killing process. Methods Bone marrow mononuclear cells （BMMNCs） derived from healthy subjects were separately cultured to generate dendritic cells （DC） and CIKs. A human mammary cancer cell line MCF-7/ADR, expressing P-gp antigen, was frozen-thawed and the lysate including P-gp antigen was obtained. The DC pulsed with or without tumor antigen lysate was co-cultured with CIK （ pulsed-DC ＋ CIK and DC ＋ CIK） , and CIK cultured alone was used as control. The cell phenotype of DC and CIK was analyzed by flow cytometry. The secretion of IL-12 and IFN-gamma was assayed by ELISA. The antitumor effect of the three CIK groups targeted at MCF-7/ADR cells expressing P-gp antigen and MCF-7 cells was detected by MTI＇. Results Pulsed-DC ＋ CIK group and DC ＋ CIK group showed a higher expression level of DC mature phenotypes than those before co-culture with CIK, with a significant difference （ P = 0. 003, P = 0. 001, respectively）. The phenotypes （CD3, CD8, CD56） of CIK in pulsed-DC ＋ CIK group and DC ＋ CIK group was higher than those in CIK group （P = 0.003, P = 0.011, respectively）. Among the three CIK groups, pulsed-DC ＋ CIK group had the highest phenotypes on CD3^＋ CD56^＋ （pulsed-DC ＋ CIK vs. DC ＋ CIK, P =0.001 ; pulsed-DC ＋ CIK vs. CIK, P 〈 0.001 ） and CD3 ^＋ CD8 ^＋ （ P = 0. 002, P = 0. 002, respectively）. Among the three groups, the pulsed- DC ＋ CIK group showed the lowest CIM5RA phenotype （ pulsed-DC ＋ CIK vs. DC ＋ CIK, P 〈 0. 001 ; pulsed-DC ＋ CIK vs. CIK, P =0.004）. Among the three groups the secretion of IL-12 and IFN-gamma had the highest level in pulsed-DC ＋ CIK group, with a value of 254 ± 14.5 pg/ml and 3100± 286 pg/ml, respectively. The antitumor killing effect on MCF-7/ADR cells had a significant difference between any two groups （ pulsed-DC ＋ CIK vs. DC ＋ CIK, P = 0.039 ; pulsed-DC ＋ CIK vs. CIK, P = 0.002 ; DC ＋ CIK vs. CIK, P = 0. 049）. The highest was in pulsed-DC ＋ CIK group and the lowest was in CIK group. The CIK group showed a significantly lower antitumor effect on MCF-7 cells than the other two groups （pulsed-DC ＋ CIK vs. CIK, P = 0. 007 ; DC ＋ CIK vs. CIK, P = 0. 048 ）, but no significant difference between the pulsed-DC ＋ CIK and DC ＋ CIK groups. Conclusion In the present study, DC and CIK cells have been successfully obtained and cultured from bone marrow mononuclear cells. After their co-culture, not only beth their specific phenotypes were increased, but also the associated cytokines were secreted. An improved antitumor killing effect and some possible specific immunocytotoxicity were observed. Our findings provided a basis for experimental and clinical research on bio-immunotherapy targeted at multi-drug resistant tumor cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2007年第10期733-737,共5页
Chinese Journal of Oncology
