论文部分内容阅读
目的:探讨重组腺病毒Ad5-CCL20联合热疗对结肠癌CT-26细胞小鼠移植瘤的抑制作用。方法:将Ad5-CCL20转染CT-26细胞,采用ELISA法和趋化实验分别检测CT-26细胞中CCL20的表达及其对DC的趋化作用。Western blotting检测热激结肠癌CT-26细胞中HSP70、GP96的表达。用热激后细胞蛋白(Heat组)对DC进行诱导,并设对照组(Control组)和阴性对照组(Unheat组),流式细胞术检测DC的表型。建立CT-26细胞移植瘤模型,设Ad5-CCL20/Heat、Heat、Ad5-CCL20、Ad5-GFP和Control组,观察各组荷瘤小鼠肿瘤生长和生存时间;采用ELISAPOT法和LDH法分别检测脾组织T淋巴细胞分泌IFN-γ的能力和对CT-26细胞的CTL杀伤活性,免疫组化法检测肿瘤组织DC浸润情况。结果:Ad5-CCL20转染CT-26细胞后可高表达CCL20,且对i DC、m DC都具有趋化活性,对i DC更为明显(P<0.05)。CT-26细胞热激后可高表达诱导型HSP70和GP96。与Control、Unheat组相比,Heat组细胞蛋白诱导后DC的CD80、CD86、CCR6和MHC-Ⅱ的表达率明显升高(均P<0.01)。与Control、Ad5-GFP、Ad5-CCL20组和Heat组相比,联合治疗组荷瘤小鼠的肿瘤抑制最为明显(均P<0.01),生存时间明显延长(均P<0.05)。联合治疗后的荷瘤小鼠脾组织T淋巴细胞的CTL活性要明显高于另外四组(均P<0.05),其瘤组织中CD11c~+ DC的阳性率也明显增高(均P<0.05)。结论:重组腺病毒Ad5-CCL20联合热疗可召募并促进DC成熟和抗原提呈,明显抑制肿瘤生长,并延长荷瘤小鼠的生存期,提示其对结肠癌有潜在的治疗作用。
Objective: To investigate the inhibitory effect of recombinant adenovirus Ad5-CCL20 combined with hyperthermia on xenografts in colon cancer CT-26 cells. Methods: CTL-26 cells were transfected with Ad5-CCL20. The expression of CCL20 and its chemotactic effect on DC were detected by ELISA and chemotaxis assay. Western blotting was used to detect the expression of HSP70 and GP96 in heat-stimulated colon cancer CT-26 cells. DCs were induced by heat-shocked cell protein (Heat group), and control group (Control group) and negative control group (Unheat group) were induced by flow cytometry. The model of CT-26 cell xenografts was established. Ad5-CCL20 / Heat, Heat, Ad5-CCL20, Ad5-GFP and Control groups were established to observe the tumor growth and survival time in each group. ELISAPOT and LDH The ability of T lymphocytes to secrete IFN-γ and CTL cytotoxic activity to CT-26 cells were detected by immunohistochemistry. Results: CCL20 was highly expressed in CT-26 cells transfected with Ad5-CCL20, and had chemotactic activity on i DC and m DC, especially on i DC (P <0.05). Heat-induced CT-26 cells can express inducible HSP70 and GP96. Compared with control group and Unheat group, the expression of CD80, CD86, CCR6 and MHC-Ⅱ in heat group were significantly increased (all P <0.01). Compared with Control group, Ad5-GFP group, Ad5-CCL20 group and Heat group, tumor-bearing mice in combination therapy group showed the most obvious tumor suppression (both P <0.01) and prolonged survival time (all P <0.05). The CTL activity of splenic T lymphocytes in the tumor-bearing mice after combined treatment was significantly higher than that in the other four groups (all P <0.05), and the positive rate of CD11c ~ + DC in the tumor-bearing mice was also significantly increased (all P <0.05) . Conclusion: Recombinant adenovirus Ad5-CCL20 combined with hyperthermia can recruit and promote DC maturation and antigen presentation, significantly inhibit tumor growth and prolong the survival of tumor-bearing mice, suggesting that it may have a potential therapeutic effect on colon cancer.