[Radix Tetrastigme Polysaccharide Promotes Antitumor Immune Response 
in Lewis Lung Cancer Mice].

Lung cancer has a high incidence and mortality rate, but the treatment of lung cancer still lacks low toxicity and efficient anti-tumor drugs. Polysaccharide from radix tetrastigme has development value in anti-tumor treatment methods. This study was to observe the effect of polysaccharide from radix tetrastigme on immune response of Lewis lung cancer mice and explore its molecular mechanism.Lewis lung cancer mouse models were established and randomly grouped. The spleen polypeptide group was intragastric with 50 mg/kg spleen polypeptide, and the radix tetrastigme polysaccharide low, medium and high dose groups were intragastric with 62.5, 125 and 250 mg/kg radix tetrastigme polysaccharide, respectively, and the model group and the control group were intragastric with equivolume normal saline. Tumor formation and metastasis were compared. Haematoxylin-eosin (HE) staining was used to observe the pathological changes of tumor cells. Macrophage phagocytosis, apoptosis, M1/M2 polarization, T cell subsets and cytokine levels in peripheral blood were detected by flow cytometry. The proliferation activity of macrophages was detected by methyl thiazolyldiphenyl tetrazolium (MTT) assay. Dendritic cell (DC) antigen presenting function was detected by chlorophenol red-β-D-galactopyranoside (CPRG) method. Tumor tissue differentiation antigen cluster 47 (CD47) mRNA and protein expression and macrophage signal regulatory protein α (SIRRP α) expression were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB).The tumor inhibition rates and anti-metastasis rates in the 3-dose radix tetrastigme polysaccharide group and the spleen polypeptide group were higher than those in the model group, and the pathological injury of tumor tissue were severer, and the positive rate of phagocytosis of ink by macrophages and the efficiency of phagocytosis of tumor cells were increased; the apoptosis rate of macrophages was decreased; the proliferation activity of macrophages, polarization ratio of macrophages to M1 type, DC antigen presenting ability, CD4+, CD4+/CD8+ levels were increased; the level of serum tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and the expression of tumor tissue CD47, macrophage SH2-containing protein tyrosine phosphatase 1 (SHP-1), SH2-containing protein tyrosine phosphatase 2 (SHP-2), and phosphorylation signal regulatory protein α (p-SIRPα) were decreased, and the differences were statistically significant (P<0.05). There were no significant differences in the above indexes between low-dose radix tetrastigme polysaccharide group and spleen polypeptide group (P>0.05), and the effects of radix tetrastigme polysaccharide were dose-dependent.Radix tetrastigme polysaccharide can inhibit tumor growth, metastasis and immune response in Lewis lung cancer mice, and its mechanism may be related to inhibiting SIRP/CD47 signaling pathway.【中文题目：三叶青多糖增强Lewis肺癌小鼠 抗肿瘤免疫反应】 【中文摘要：背景与目的 肺癌具有高致病率和高致死率，但肺癌治疗仍缺乏低毒高效的抗肿瘤药物。三叶青多糖在抗肿瘤治疗方面具有开发价值。本文旨在观察三叶青多糖对Lewis肺癌小鼠免疫反应的影响，并探讨其分子机制。方法 建立Lewis肺癌小鼠模型并采用随机数字表法进行分组。脾多肽组灌胃50 mg/kg脾多肽，三叶青多糖低、中、高剂量组分别灌胃62.5、125、250 mg/kg三叶青多糖，模型组和对照组灌胃等容积生理盐水。比较肿瘤形成、转移情况；苏木素-伊红（haematoxylin-eosin, HE）染色观察肿瘤细胞病理学变化；流式细胞术检测巨噬细胞吞噬能力、凋亡水平、M1/M2型极化水平及外周血T细胞亚群和细胞因子水平；噻唑蓝溴化四唑（methyl thiazolyldiphenyl tetrazolium, MTT）法检测巨噬细胞增殖活性；β-D-吡喃半乳糖苷氯酚红（chlorophenol red-β-D-galactopyranoside, CPRG）法检测树突状细胞（dendritic cell, DC）抗原提呈功能；实时荧光定量聚合酶链反应（real time quantitative polymerase chain reaction, RT-qPCR）和蛋白质免疫印迹法（Western blot, WB）检测肿瘤组织分化抗原簇47（cluster of differentiation 47, CD47）信使核糖核酸（messenger ribonucleic acid, mRNA）和蛋白表达及巨噬细胞中信号调节蛋白α（signal regulatory protein α, SIRPα）表达。结果 三叶青多糖3个剂量组和脾多肽组抑瘤率和抗转移率高于模型组，肿瘤组织病理损伤严重，巨噬细胞吞噬墨汁阳性率和吞噬肿瘤细胞效率升高，巨噬细胞凋亡率下降，巨噬细胞增殖活性、巨噬细胞向M1型极化比例、DC抗原提呈能力、CD4+、CD4+/CD8+升高，血清肿瘤坏死因子α（tumor necrosis factor α, TNF-α）、白介素-1β（interleukin-1β, IL-1β）、肿瘤组织CD47、巨噬细胞蛋白酪氨酸磷酸酶1（SH2-containing protein tyrosine phosphatase 1, SHP-1）、蛋白酪氨酸磷酸酶2（SH2-containing protein tyrosine phosphatase 2, SHP-2）及磷酸化信号调节蛋白α（phosphorylation signal regulatory protein α, p-SIRPα）表达下降，差异均有统计学意义（P<0.05）。上述指标三叶青多糖低剂量组与脾多肽组的差异均无统计学意义（P>0.05），且三叶青多糖的作用均呈剂量依赖性。结论 三叶青多糖有抗Lewis肺癌小鼠肿瘤生长和转移及免疫反应的作用，其机制可能与抑制SIRP/CD47信号通路有关。 】 【中文关键词：三叶青多糖；信号调节蛋白α；分化抗原簇47；肺肿瘤；免疫反应】.