Zhang You-Shun, Yuan Fang-Jun, Jia Guo-Feng, Zhang Ji-Fa, Hu Li-Yi, Huang Ling, Wang Ju, Dai Zong-Qing
Institute of Liver Surgery, Dongfeng Hospital of YunYang Medical College, Shiyan 442008, Hubei Province, China.
World J Gastroenterol. 2005 Jun 14;11(22):3339-45. doi: 10.3748/wjg.v11.i22.3339.
To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo.
A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2,000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-gamma, monoclonal anti-CD3 antibody, rhIL-1alpha as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68.
A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5 x 10(10) of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry.
A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.
探讨原发性肝癌（HCC）术后患者的细胞因子诱导的杀伤（CIK）细胞对HCC多药耐药（MDR）细胞的体内外细胞毒性。
通过在含有浓度从10至2000 nmol/L递增阿霉素的完全RPMI 1640培养基中培养人HCC细胞系Bel-7402建立耐药细胞系。用重组人干扰素-γ（rhIFN-γ）、抗CD3单克隆抗体、重组人白细胞介素-1α（rhIL-1α）以及重组人白细胞介素-2（rhIL-2）诱导外周血单个核细胞获得CIK细胞，并将其加入培养体系。以Bel-7402/R作为靶（T）细胞，CIK细胞作为效应（E）细胞，检测HCC患者CIK细胞的细胞毒性。采用MTT法进行细胞毒性试验并检测。在体内试验中，将CIK细胞输注给HCC患者。获取患者的肿瘤标本并进行免疫组织化学检测CD3、CD45、CD45RO以及CD68。
建立了一株MDR1 HCC细胞系Bel-7402/R。逆转录聚合酶链反应（RT-PCR）显示其MDR1 mRNA过表达；P-糖蛋白表达从亲代细胞的1.32%增加至54%。CIK细胞在平均培养3周后活力旺盛地扩增了70多倍，CD3+CD56+增加了600多倍。HCC患者的CIK细胞对Bel-7402/R的细胞毒性约为50%，对L-02的细胞毒性低于10%（t = 8.87，P<0.01），与正常个体的CIK细胞相同。17例患者每人接受了1 - 5×10¹⁰个CIK细胞输注。未观察到副作用。CIK治疗后，肿瘤组织形成结节，肝癌组织中有大量淋巴细胞浸润，免疫组织化学显示CD3、CD45、CD45RO和CD68显著增加。
建立了一株稳定的MDR1 HCC细胞系，该细胞系可从液氮中复苏，HCC患者的CIK细胞对MDR HCC细胞具有强大的细胞毒性。CIK过继性免疫治疗安全且无副作用。接受者对肿瘤的免疫力明显提高。CIK治疗可能是HCC术后患者预防复发的较好选择，尤其是当肿瘤已产生耐药性时。
