Jiao Hongliang, Yang Bo, Guan Fangxia, Li Jianbin, Shan Hong, Song Laijun, Hu Xiang, Liang Shuo, Du Ying, Jiang Chao
Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Neurol Res. 2011 May;33(4):405-14. doi: 10.1179/016164110X12816242542490.
It is difficult for the treatment of neurologic tumors with current therapies, including glioma. Despite the advances in cancer therapeutics, the outcomes in these patients remain poor and, therefore, new modalities are required. Recent findings have demonstrated that human umbilical cord blood mesenchymal stem cells (UCB-MSCs) have the potential to inhibit glioma cell growth in vitro. Caspase-3 plays a critical role as an executioner of apoptosis and the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. CD133 (+) glioma displays a strong resistance to chemotherapy. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are important cytokines in the generation of antitumor immunity.
UCB-MSCs were harvested by density gradient separation. Green fluorescence protein stable C6 cells were established. Cytotoxicity was detected by visual survival cell assay, and caspase-3 activity was assessed using immunohistochemistry staining and western blot. Flow cytometry was used to test CD133 positive C6 cells. The concentrations of IL-2 and IFN-gamma proteins secreted from UCB-MSCs were then quantified using enzyme-linked immunosorbent assay. The cytotoxicities of IL-2 alone, IFN-gamma alone, and combination of IL-2 and IFN-gamma were compared against malignant glioma cells.
We noted a significant cytotoxicity of UCB-MSCs for malignant glioma cells. In addition, the toxicity of mixed UCB-MSCs was significantly higher than that of single UCB-MSCs for C6 glioma cells. Capase-3 levels in UCB-MSCs treatment at an effector/target (E/T) ratio of (5+5):1 were higher than those at an E/T ratio of 10:1. On the contrary, there was no change in the protein expression of capase-3 at an E/T ratio of 0:1. Immunohistochemistry staining and western blot revealed that the expression of capase-3 was higher in mixed UCB-MSCs treatment, when compared with single UCB-MSCs. We identified reductions of approximately 39 and 73% in the number of CD133 positive C6 cells treated with the single (10:1) and the mixed [(5+5):1] UCB-MSCs respectively as compared to the control group (0:1) in transwell inserts. However, the mixed UCB-MSCs secreted more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma might prove more cytotoxic on target cells than IL-2 alone and IFN-gamma alone.
The data collected herein confirm for the first time that the mixed UCB-MSCs were shown to have more cytotoxic effects than the single UCB-MSCs through increasing the expression of caspase-3 and decreasing the expression of CD133 in C6 glioma cells. In addition, the mixed UCB-MSCs secrete more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma was shown to have more cytotoxic effects than IL-2 alone and IFN-gamma alone. These results demonstrate that the mixed UCB-MSCs are a potential new therapeutic agent for glioma.
包括胶质瘤在内的神经肿瘤目前的治疗方法存在困难。尽管癌症治疗取得了进展，但这些患者的治疗效果仍然很差，因此需要新的治疗方式。最近的研究发现表明，人脐带血间充质干细胞（UCB - MSCs）在体外具有抑制胶质瘤细胞生长的潜力。半胱天冬酶 - 3作为凋亡的执行者发挥着关键作用，其表达水平决定了癌细胞系中的凋亡程度。CD133(+)胶质瘤对化疗表现出强烈抗性。白细胞介素 - 2（IL - 2）和干扰素 - γ（IFN - γ）是抗肿瘤免疫产生中的重要细胞因子。
通过密度梯度分离法收集UCB - MSCs。建立绿色荧光蛋白稳定表达的C6细胞。通过可视存活细胞测定法检测细胞毒性，并使用免疫组织化学染色和蛋白质印迹法评估半胱天冬酶 - 3活性。采用流式细胞术检测CD133阳性C6细胞。然后使用酶联免疫吸附测定法定量UCB - MSCs分泌的IL - 2和IFN - γ蛋白浓度。将单独的IL - 2、单独的IFN - γ以及IL - 2与IFN - γ联合使用时对恶性胶质瘤细胞的细胞毒性进行比较。
我们注意到UCB - MSCs对恶性胶质瘤细胞具有显著的细胞毒性。此外，混合的UCB - MSCs对C6胶质瘤细胞的毒性明显高于单一的UCB - MSCs。效应细胞与靶细胞（E/T）比例为(5 + 5):1时，UCB - MSCs处理组中的半胱天冬酶 - 3水平高于E/T比例为10:1时。相反，E/T比例为0:1时，半胱天冬酶 - 3的蛋白表达没有变化。免疫组织化学染色和蛋白质印迹显示，与单一UCB - MSCs相比，混合UCB - MSCs处理时半胱天冬酶 - 3的表达更高。在transwell小室中，与对照组（0:1）相比，用单一（10:1）和混合[(5 + 5):1] UCB - MSCs处理的CD133阳性C6细胞数量分别减少了约39%和73%。然而，混合UCB - MSCs比单一UCB - MSCs分泌更多与免疫反应相关的蛋白（IL - 2和IFN - γ）。IL - 2与IFN - γ联合使用对靶细胞的细胞毒性可能比单独使用IL - 2和单独使用IFN - γ更大。
本文收集的数据首次证实，混合的UCB - MSCs通过增加C6胶质瘤细胞中半胱天冬酶 - 3的表达和降低CD133的表达，显示出比单一UCB - MSCs更强的细胞毒性作用。此外，混合UCB - MSCs比单一UCB - MSCs分泌更多与免疫反应相关的蛋白（IL - 2和IFN - γ）。IL - 2与IFN - γ联合使用显示出比单独使用IL - 2和单独使用IFN - γ更强的细胞毒性作用。这些结果表明，混合UCB - MSCs是一种潜在的胶质瘤新治疗药物。
