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目的探讨斯氏艾美球虫(Eimeria stiedai)可溶性蛋白(EsSP)对荷瘤小鼠肿瘤生长、生存率和免疫状态的影响。方法建立小鼠皮下结肠癌(CT26)肿瘤模型,确定最小100%致瘤量肿瘤细胞数。取10~8个斯氏艾美球虫孢子化卵囊,采用超声波间断乳化制备EsSP。105只雄性BALB/c小鼠按随机数表法均分为7组(15只/组),每组小鼠于右侧腋窝皮下接种CT26细胞5×10~5个,其中6个实验组(A~F组)小鼠分别腹腔注射100.00、50.00、10.00、1.00、0.10、0.01μg/d EsSP,1次/d×5 d,对照组腹腔注射等量PBS。于接种后第7、11、13、15、17、19、21、23天测量肿瘤直径,计算相对肿瘤体积和相对肿瘤增殖率(T/C)。接种后第25天每组各处死5只小鼠,称量瘤重,计算抑瘤率。采眼球血,分离小鼠外周血淋巴细胞,MTS比色法检测EsSP对荷瘤小鼠淋巴细胞增殖能力的影响,计算刺激指数(SI);流式细胞术检测外周血CD4~+/CD8~+T淋巴细胞比值变化。记录各组荷瘤小鼠死亡时间和死亡数量,共观察80 d。各组间差异性比较采用单因素方差分析。结果最小100%致瘤量肿瘤细胞数为5×10~5个。接种结肠癌细胞后第23天,A、B、C组的肿瘤体积为(435.2±41.1)、(366.3±29.2)、(460.2±28.5)mm~3,较E、F和对照组的(761.2±33.2)、(810.4±38.4)、(865.2±35.3)mm~3生长缓慢(P<0.05);A、B、C组的T/C分别为(39.0±6.7)%、(33.3±8.9)%、(35.0±8.1)%,均<40%。B组小鼠瘤重为(1.109±0.432)g,低于对照组的(1.946±0.289)g(P<0.05),抑瘤率最高,为(43.0±14.6)%。MTS比色法检测结果显示,B、C组小鼠SI分别为1.75±0.15、1.70±0.32,高于对照组的1.38±0.18(P<0.05)。流式细胞术检测结果显示,A、B、C组小鼠外周血中CD4~+/CD8~+T淋巴细胞亚群的比值分别为1.58±0.24、1.74±0.22、1.61±0.16,与对照组的1.34±0.15比较,差异均有统计学意义(P<0.01或P<0.05)。荷瘤小鼠生存率结果显示,至第80天,B、C组各存活5只小鼠,高于对照组的2只(P<0.05)。结论 EsSP能够抑制结肠癌皮下肿瘤的生长,提高荷瘤小鼠生存率,改变肿瘤诱导的免疫抑制状态,增强小鼠抗肿瘤免疫反应。
Objective To investigate the effect of Eimeria stomatica soluble protein (EsSP) on tumor growth, survival rate and immune status in tumor-bearing mice. Methods Mouse subcutaneous colon cancer (CT26) tumor model was established to determine the minimum number of tumorigenic tumor cells. Take 10 to 8 Escherichia coli sporulated oocysts, prepared by intermittent ultrasonic emulsification EsSP. 105 male BALB / c mice were randomly divided into 7 groups (15 mice / group) according to random number table. Each group of mice were inoculated subcutaneously with 5 × 10 ~ 5 CT26 cells on the right armpit. Six experimental groups Groups A to F) were injected intraperitoneally with EsSP (100.00, 50.00, 1.00, 1.00, 0.10 and 0.01 μg / d, respectively) once a day for 5 days. Tumor diameter was measured on days 7, 11, 13, 15, 17, 19, 21 and 23 after inoculation, and the relative tumor volume and relative tumor growth rate (T / C) were calculated. On the 25th day after inoculation, 5 mice were sacrificed in each group, the tumor weights were weighed, and the tumor inhibition rate was calculated. The eyeball blood was collected to separate the peripheral blood lymphocytes of mice. MTS colorimetric assay was used to detect the effect of EsSP on the proliferation of tumor-bearing mice and the stimulation index (SI) was calculated. Flow cytometry was used to detect the percentage of CD4 ~ + / CD8 ~ + T lymphocyte ratio changes. The death time and the number of death in each group of tumor-bearing mice were recorded and observed for 80 days. The differences between groups were compared using one-way analysis of variance. Results The minimum tumorigenicity of 100% tumor cells was 5 × 10 ~ 5. The tumor volume in groups A, B and C was (435.2 ± 41.1), (366.3 ± 29.2) and (460.2 ± 28.5) mm ~ 3 on day 23 after inoculation of colon cancer cells, (P <0.05). The T / C of group A, B and C were (39.0 ± 6.7)% and (33.3 ± 8.9)%, respectively %, (35.0 ± 8.1)%, both <40%. The tumor weight in group B was (1.109 ± 0.432) g, which was lower than that in control group (1.946 ± 0.289) g (P <0.05). The tumor inhibition rate was the highest (43.0 ± 14.6)% in group B. The results of MTS assay showed that SI in group B and C were 1.75 ± 0.15 and 1.70 ± 0.32, respectively, which was higher than that in control group (1.38 ± 0.18, P <0.05). The results of flow cytometry showed that the ratio of CD4 ~ + / CD8 ~ + T lymphocyte subsets in peripheral blood of mice in groups A, B and C were 1.58 ± 0.24, 1.74 ± 0.22 and 1.61 ± 0.16, respectively. Compared with the control group Of 1.34 ± 0.15, the differences were statistically significant (P <0.01 or P <0.05). The survival rate of tumor-bearing mice showed that by the 80th day, 5 mice survived in groups B and C, which was higher than that in control group (P <0.05). Conclusion EsSP can inhibit the growth of colon cancer subcutaneous tumors, improve the survival rate of tumor-bearing mice, change the state of tumor-induced immunosuppression and enhance anti-tumor immune response in mice.