Lgr5蛋白激活树突状细胞诱导抗原特异性细胞毒T淋巴细胞治疗结肠癌的实验

目的 探讨富含亮氨酸重复序列的G蛋白耦联受体5(Lgr5）蛋白激活树突状细胞(DCs),诱导产生CD8+ 细胞毒T淋巴细胞（CTL）进行结肠癌免疫治疗的效果。 方法 利用Lgr5蛋白诱导DCs成熟，同时检测DCs表面标记物和白细胞介素（IL)-10与IL-12表达量的变化，随后通过Lgr5-DC诱导Lgr5抗原特异性CD8⁺ CTL，并检测Lgr5-DC-CD8⁺ CTL对正常结肠上皮细胞CCD-18Co和结肠癌细胞HT29的作用，同时检测干扰素（IFN）-γ释放量。然后进一步检测Lgr5-DC-CD8⁺ CTL对BALB/C-nu/nu小鼠结肠癌的抑制情况，并通过组织染色观察治疗后肿瘤组织的变化。 结果 与PBS刺激相比，Lgr5蛋白刺激能够显著上调DCs表面标记物DC80、DC83、DC86和HLA-DR水平，依次达到3.29、3.06、2.90和6.93倍；同时Lgr5蛋白刺激显著促进IL-12的释放和显著减少IL-10的分泌(P＜0.05)。Lgr5-DC-CD8⁺ CTL和DC-CD8⁺ CTL均导致少量CCD-18Co细胞杀伤(P＞0.05)， 而Lgr5-DC-CD8⁺ CTL对HT29细胞的杀伤率是DC-CD8+ CTL的4.40倍(P＜0.05)。动物实验表明，BALB/C-nu/nu结肠癌移植鼠经Lgr5-DC-CD8⁺ CTL治疗后，肿瘤体积比显著低于PBS组和DC-CD8⁺ CTL组，依次达到0.25和0.24倍(P＜0.05)。组织染色显示 Lgr5-DC-CD8⁺CTL处理导致明显的肿瘤组织病理学改变，同时BAX表达升高。 结论 Lgr5蛋白促进DCs成熟并诱导产生Lgr5抗原特异性CD8⁺ CTL，Lgr5-DC-CD8⁺ CTL能够高效的杀伤肿瘤细胞并延迟肿瘤生长。

Experimental immunotherapy study on colon cancer by lgr5 activated dendritic cells to induce antigen-specific CD8 + cytotoxic T lymphocytes

Objective To observe the immunotherapy effect on colon cancer by leucine-rich repeat-containing Gprotein coupled receptor 5 (Lgr5) activated by dexxeperimental basis for colon cancer immunotherapy.Methods After dendritic cells (DCs) maturation induced by Lgr5,surface molecules,interleukin (IL)-12,and IL-10 of DCs were detected.Subsequently,Lgr5 antigen specific CDg + cytotoxic T lymphocyte (CTL) was induced by Lgr5-DC.The killing effect and interferon (IFN)-γ of Lgr5-DC-CD8 + CTL on normal colonic epithelial cells CCD-18Co and colon cancer cell HT29 were tested.After Lgr5-DC-CD8 + CTL treatment,tumor volume in BALB/C-nu/nu mice was detected.The morphology of tumor tissue after treatment was observed by tissue staining.Results Compared with PBS,Lgr5 protein stimulation significantly increased surface markers DC80,DC83,DC86 and HLA-DR levels,and up to 3.29,3.06,2.90 and 6.93 times (P ＜ 0.05) respectively.Lgr5 stimulation significantly stimulated the release of IL-12 and significantly reduced the secretion of IL-10 (P ＜ 0.05).DC-CD8 + CTL and Lgr5-DC-CD8 + CTL.Both resulted in a small amount of CCD-18Co cell killing (P ＞ 0.05),but the killing rate of Lgr5-DC-CD8 + CTL on HT29 cells was 4.40 times as much as that of DC-CD8 + CTL.Tissue staining showed that Lgr5-DC-CD8 + CTL treatment resulted in significant pathological changes and BAX expression in tumor tissues.Conclusion Lgr5 protein stimulated the maturation of DCs cells that induced the production of Lgr5 antigen specific CD8 + CTL.Lgr5-DC-CD8 + CTL can effectively kill tumor cells and delay the growth of tumor.