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

马刚 杨庆强* 何兴状

doi:10.16098/j.issn.0529-1356.2017.04.010

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解剖学报 ›› 2017, Vol. 48 ›› Issue (4) : 428-433. DOI: 10.16098/j.issn.0529-1356.2017.04.010

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

马刚^1,2 杨庆强^1* 何兴状³

作者信息 +

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

MA Gang^1,2 YANG Qing-qiang^1* HE Xing-zhuang³

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摘要

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。

Abstract

Objective To observe the immunotherapy effect on colon cancer by leucine-rich repeat-containing G-protein 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 CD8⁺ 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.

关键词

富含壳氨酸重复序列的G蛋白耦联受体 / 树突状细胞 / 细胞毒T淋巴细胞 / 结肠癌 / 免疫治疗 / 小鼠

Key words

Leucine-rich repeat-containing G-protein collpled receptor 5

/ Dendritic cell / Cytotoxic T lymphocyte / Colon cancer / Immunotherapy / Mouse

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马刚杨庆强何兴状. 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[J]. 解剖学报. 2017, 48(4): 428-433 https://doi.org/10.16098/j.issn.0529-1356.2017.04.010

MA Gang YANG Qing-qiang HE Xing-zhuang. Experimental immunotherapy study on colon cancer by lgr5 activated dendritic cells to induce antigen-specific CD8+ cytotoxic T lymphocytes[J]. Acta Anatomica Sinica. 2017, 48(4): 428-433 https://doi.org/10.16098/j.issn.0529-1356.2017.04.010

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