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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-DCCD8~+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。
Objective To investigate the effect of Lgr5-rich dendritic cells (DCs) enriched in leucine repeats on the immunotherapy of colon cancer induced by CD8 + cytotoxic T lymphocytes (CTLs) . Methods The Lgr5 protein was used to induce the maturation of DCs, and the changes of the surface markers of DCs and the expressions of IL-10 and IL-12 were detected. Lgr5 antigen-specific CD8 + CTL was induced by Lgr5-DC and Lgr5 -DC-CD8 ~ + CTL on normal colon epithelial cells CCD-18Co and colon cancer cells HT29, while interferon (IFN) -γ release was detected. The inhibitory effect of Lgr5-DC-CD8 ~ + CTL on colon cancer in BALB / C-nu / nu mice was further detected, and the changes of tumor tissues after treatment were observed by tissue staining. Results Compared with PBS stimulation, Lgr5 stimulation significantly up-regulated DCs surface markers such as DC80, DC83, DC86 and HLA-DR, reaching 3.29, 3.06, 2.90 and 6.93 times, respectively. Simultaneously Lgr5 stimulation significantly promoted the release of IL-12 And significantly decreased IL-10 secretion (P <0.05). Lgr5-DC-CD8 + CTL and DC-CD8 + CTL all caused a small amount of CCD-18Co cell killing (P> 0.05), while the lethal dose of Lgr5-DCCD8 + CTL to HT29 cells was 4.40 Times (P <0.05). Animal experiments showed that the tumor volume ratio of BALB / C-nu / nu colon cancer transplanted mice was significantly lower than that of PBS group and DC-CD8 ~ + CTL group after Lgr5-DC-CD8 + CTL treatment, reaching 0.25 and 0.24 times P <0.05). Tissue staining showed that Lgr5-DC-CD8 ~ + CTL treatment resulted in significant tumor histopathological changes, while BAX expression increased. Conclusion Lgr5 protein can promote the maturation of DCs and induce the production of Lgr5 antigen - specific CD8 ~ + CTL. Lgr5-DC-CD8 ~ + CTL can effectively kill tumor cells and delay tumor growth.