Sun E, Nian X, Liu C, Fan X, Han R
Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin, China.
Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China.
Genet Mol Res. 2015 Apr 15;14(2):3436-49. doi: 10.4238/2015.April.15.7.
We constructed recombinant Bacille Calmette-Guérin (rBCG) that secreted human interferon alpha 2b (hIFNα-2b), and investigated its antitumor effects on bladder cancer cells in vitro. The recombinant plasmid phIFN-α-2b was constructed using pMAO-4 and transformed into BCG. The supernatant was collected at various times and IFN-γ, interleukin (IL)-12, and tumor necrosis factor (TNF)-α were detected using an enzyme-linked immunosorbent assay. EJ cells were cultivated for 24, 48, and 72 h, together with rBCG, wild-type BCG (wBCG), or wBCG+IFN-α-2b. rBCG capable of secreting cytokine IFNα-2b was constructed. On the 4th day of culture, the IFNα-2b secreted by rBCG reached a maximum. wBCG and rBCG showed no significant difference on cell growth rate over 7 days of incubation in 7H9 medium. wBCG and rBCG were both positive for acid-fast staining, and showed mycobacterial characteristics of intercellular connection in clusters with no clear abnormalities. Higher levels of IFN-γ, TNF-α, and IL-12 were induced by rBCG compared with wBCG or MAO4-rBCG (P < 0.05). rBCG may induce lymphocyte proliferation; the proliferation ratio was higher than those induced by wBCG and wBCG+IFN. rBCG had direct anti-proliferative effects on EJ cells. An MTT assay showed that rBCG inhibited the proliferation of bladder cancer cells and had more activity compared with wBCG (P < 0.05). The highest anti-tumor activity of lymphocytes was stimulated by rBCG (20.31-51.22%). rBCG-IFNα-2b induces enhanced cytotoxicity against bladder cancer cells in vitro and may be used as an alternative to BCG for bladder cancer patients.
我们构建了分泌人干扰素α2b（hIFNα - 2b）的重组卡介苗（rBCG），并在体外研究了其对膀胱癌细胞的抗肿瘤作用。使用pMAO - 4构建重组质粒phIFN - α - 2b，并将其转化到卡介苗中。在不同时间收集上清液，使用酶联免疫吸附测定法检测干扰素γ（IFN - γ）、白细胞介素（IL）- 12和肿瘤坏死因子（TNF）- α。将EJ细胞与rBCG、野生型卡介苗（wBCG）或wBCG + IFN - α - 2b一起培养24、48和72小时。构建了能够分泌细胞因子IFNα - 2b的rBCG。在培养的第4天，rBCG分泌的IFNα - 2b达到最大值。在7H9培养基中孵育7天，wBCG和rBCG在细胞生长速率上无显著差异。wBCG和rBCG的抗酸染色均为阳性，且显示出细胞间呈簇状连接的分枝杆菌特征，无明显异常。与wBCG或MAO4 - rBCG相比，rBCG诱导产生更高水平的IFN - γ、TNF - α和IL - 12（P < 0.05）。rBCG可能诱导淋巴细胞增殖；增殖率高于wBCG和wBCG + IFN诱导的增殖率。rBCG对EJ细胞有直接的抗增殖作用。MTT试验表明，rBCG抑制膀胱癌细胞的增殖，且与wBCG相比具有更强的活性（P < 0.05）。rBCG刺激淋巴细胞产生的抗肿瘤活性最高（20.31 - 51.22%）。rBCG - IFNα - 2b在体外诱导对膀胱癌细胞的细胞毒性增强，可作为膀胱癌患者卡介苗的替代物。
