干酪乳杆菌对二甲基苯蒽诱发大鼠乳腺肿瘤的抑制作用及其机制研究

发布时间：2018-08-24 10:56

【摘要】：目的:本实验通过观察干酪乳杆菌(Lactobacillus casei)对二甲基苯蒽(DMBA)诱发乳腺癌大鼠的血清中细胞因子和外周血中自然杀伤(NK)细胞活性、T淋巴细胞亚群分布的影响来评价干酪乳杆菌对乳腺癌大鼠免疫功能的调节。通过观察干酪乳杆菌对二甲基苯蒽诱发乳腺癌大鼠的肠道菌群和肠道连接蛋白表达的影响来评价干酪乳杆菌对乳腺癌大鼠肠道屏障功能的调节。为进一步阐述干酪乳杆菌对二甲基苯蒽诱发的大鼠乳腺肿瘤的抑制作用机制提供实验依据。方法:1.干酪乳杆菌对二甲基苯蒽诱发乳腺肿瘤的抑制作用及其免疫功能的影响。(1)动物分组及乳腺癌模型建立:60只雌性SD大鼠适应性喂养一周后,按体质量随机分为正常对照组、模型组、干酪乳杆菌剂量1组和干酪乳杆菌剂量2组。模型组及干酪乳杆菌剂量1组和2组大鼠右侧臀部皮下一次性注射100 mg/kg DMBA建立乳腺癌模型。干酪乳杆菌剂量1组和2组分别灌胃给予4 m L/(kg·d)和8 m L/(kg·d)干酪乳杆菌(1×108 CFU/m L),正常对照组和模型组均给予5 m L/(kg·d)大豆油灌胃。每天1次,持续16周后处死大鼠,腹主动脉取血并留取胸腺,脾脏,小肠组织。(2)计算各组大鼠乳腺癌发生率、潜伏期、平均瘤重、抑瘤率及脏器指数。(3)流式细胞术检测大鼠外周血中NK细胞活性和T淋巴细胞亚群分布。(4)酶联免疫吸附实验(ELISA)检测血清中细胞因子白介素-4(IL-4)、IL-6、IL-10、IL-12、干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)的水平。2.干酪乳杆菌对二甲基苯蒽诱导乳腺癌大鼠肠道菌群和肠道屏障功能的影响。(1)实时荧光定量PCR(q PCR)检测大鼠肠道菌群的变化。(2)HE染色观察大鼠空肠组织病理学改变。(3)ELISA检测大鼠血清中D-乳酸(D-LA)水平。(4)Western Blotting检测大鼠空肠组织紧密连接蛋白occludin和ZO-1的表达水平。结果:1.干酪乳杆菌对二甲基苯蒽诱发乳腺肿瘤的抑制作用及其免疫功能的影响。(1)正常对照组大鼠无肿瘤发生,而模型组、干酪乳杆菌剂量1组和2组均有肿瘤发生。与模型组比较,干酪乳杆菌剂量2组大鼠肿瘤潜伏期延长,肿瘤发生率和平均瘤质量降低(P0.05);抑瘤率达到41.2%;且该组胸腺指数显著升高(P0.05);(2)流式细胞术结果显示,与模型组比较,干酪乳杆菌剂量2组大鼠TCRαβ+CD161a+NK细胞百分比、CD3+CD8+T细胞百分比均显著升高(P0.05);而血中CD3+Foxp3+细胞百分比明显下降(P0.05)。(3)与正常对照组比较,模型组IL-4水平明显降低,而IL-6、IL-12和TNF-α水平显著升高(P0.05);与模型组比较,干酪乳杆菌剂量2组血清IL-4、IL-10浓度显著增高,IL-6、IL-12浓度显著下降(P0.05);干酪乳杆菌剂量1组和2组TNF-α浓度均显著下降(P0.05)。2.干酪乳杆菌对二甲基苯蒽诱导乳腺癌大鼠肠道菌群和肠道屏障功能的影响。(1)q PCR结果显示,与正常对照组比较,模型组双歧杆菌、乳酸杆菌和粪肠球菌数量均下降,其中双歧杆菌和粪肠球菌数量下降有统计学意义(P0.05);干酪乳杆菌剂量1组和2组双歧杆菌数量均比模型组显著升高(P0.05),而大肠杆菌的数量有所降低。(2)HE染色结果显示,与模型组比较,干酪乳杆菌剂量1组和2组大鼠空肠内绒毛和隐窝的形态明显完整,结构清晰,绒毛的损伤程度较小。但与正常组比较,干酪乳杆菌剂量1组和2组小肠黏膜厚度减少,绒毛的排列也较为凌乱,绒毛尖端有水肿的现象。(3)ELISA结果显示,与正常对照组比较,模型组D-LA浓度显著升高,而与模型组比较,干酪乳杆菌剂量2组D-LA浓度显著下降(P0.05)。(4)Western Blotting结果显示,与正常对照组比较,模型组紧密连接蛋白occludin和ZO-1的表达均显著下降,而干酪乳杆菌剂量2组均比模型组显著升高(P0.05)。结论:1.干酪乳杆菌对大鼠乳腺肿瘤的生长具有一定的抑制作用。2.干酪乳杆菌可调节CD4+、CD8+T细胞、NK细胞及调节性T细胞等免疫细胞分布,改善炎性相关细胞因子水平,提高机体免疫调节作用。3.干酪乳杆菌可改善肠道菌群的分布,降低血清中D-LA浓度、增加肠道紧密连接蛋白occludin、ZO-1的表达,提高机体的肠道屏障功能。
[Abstract]:Objective: To evaluate the effect of Lactobacillus casei on the immune function of breast cancer rats by observing the activity of cytokines in serum and natural killer (NK) cells in peripheral blood and the distribution of T lymphocyte subsets in serum of breast cancer rats induced by Lactobacillus casei. To evaluate the effect of Lactobacillus casei on intestinal barrier function in rats with breast cancer induced by p-dimethylbenzoanthracene. To further elucidate the inhibitory mechanism of Lactobacillus casei on mammary tumor induced by p-dimethylbenzoanthracene. Methods: 1. Cheese milk. (1) Animal grouping and establishment of breast cancer model: 60 female SD rats were randomly divided into normal control group, model group, Lactobacillus casei dosage group 1 and Lactobacillus casei dosage group 2 according to body weight after one week of adaptive feeding. Breast cancer models were established by subcutaneous injection of 100 mg/kg DMBA into the right buttocks of rats in group 1 and group 2. Lactobacillus casei dosage group 1 and group 2 were given Lactobacillus casei (4 m L/(kg.d) and 8 m L/(kg.d) respectively by intragastric administration. Normal control group and model group were given 5 m L/(kg.d) soybean oil once a day for 16 weeks. Blood samples were taken from the abdominal aorta and thymus, spleen and small intestine tissues were taken from the dead rats. (2) The incidence of breast cancer, latency, average tumor weight, tumor inhibition rate and organ index were calculated. (3) The activity of NK cells and the distribution of T lymphocyte subsets in the peripheral blood of rats were detected by flow cytometry. (4) Serum cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Levels of interleukin-4 (IL-4), IL-6, IL-10, IL-12, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the intestinal flora and intestinal barrier function of breast cancer rats induced by dimethylbenzoanthracene. (1) Changes of intestinal flora in rats were detected by real-time fluorescence quantitative PCR (q PCR). (2) HE staining was used to observe jejunal histopathy in rats. Physical changes. (3) The level of D-lactic acid (D-LA) in serum was detected by ELISA. (4) The expression of occludin and ZO-1 in jejunum was detected by Western Blotting. Results: 1. The inhibitory effect of Lactobacillus casei on dimethylbenzoanthracene-induced breast tumor and its immune function were observed. (1) There was no tumor in normal control group. Compared with the model group, the tumor incubation period was prolonged, the tumor incidence and the average tumor mass were decreased (P 0.05), the tumor inhibition rate was 41.2%, and the thymus index was significantly increased (P 0.05). (2) Compared with the model group, the results of flow cytometry showed that the tumor incubation period was prolonged, the tumor incidence and the average tumor mass were decreased (P 0.05). The percentage of TCR alpha beta+CD161a+NK cells and the percentage of CD3+CD8+T cells were significantly increased (P 0.05), while the percentage of CD3+Foxp3+ cells in blood was significantly decreased (P 0.05). (3) Compared with the normal control group, the level of IL-4 in the model group was significantly decreased, while the levels of IL-6, IL-12 and TNF-alpha were significantly increased (P 0.05). The serum levels of IL-4, IL-10, IL-6 and IL-12 in the two groups were significantly higher than those in the control group (P The number of bifidobacteria, Lactobacillus and Enterococcus faecalis in model group decreased, and the number of bifidobacteria and Enterococcus faecalis decreased significantly (P 0.05); the number of bifidobacteria in group 1 and group 2 of Lactobacillus casei was significantly higher than that in model group (P 0.05), but the number of Escherichia coli was decreased. (2) HE staining results showed that the number of bifidobacteria and Enter Compared with the normal group, the thickness of intestinal mucosa, the arrangement of villi and the edema of villi tips in groups 1 and 2 of Lactobacillus casei dosage were reduced, and the edema of villi tips was found in the group 1 and 2 of Lactobacillus casei dosage. Compared with the normal control group, the D-LA concentration in the model group was significantly increased, while the D-LA concentration in the Lactobacillus casei dosage group was significantly decreased (P 0.05). (4) Compared with the normal control group, the expression of occludin and ZO-1 in the model group were significantly decreased, while the expression of occludin and ZO-1 in the Lactobacillus casei dosage group were significantly lower than that in the model group. Conclusion: 1. Lactobacillus casei can inhibit the growth of mammary gland tumor in rats. 2. Lactobacillus casei can regulate the distribution of CD4 +, CD8 + T cells, NK cells and regulatory T cells, improve the levels of inflammatory cytokines, improve the immune regulation of the body. 3. Lactobacillus casei can improve the intestinal flora. The distribution of D-LA decreased the concentration of D-LA in serum, increased the expression of intestinal tight junction protein occludin and ZO-1, and enhanced the intestinal barrier function.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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