【摘要】：目的:本實(shí)驗(yàn)通過觀察干酪乳桿菌(Lactobacillus casei)對二甲基苯蒽(DMBA)誘發(fā)乳腺癌大鼠的血清中細(xì)胞因子和外周血中自然殺傷(NK)細(xì)胞活性、T淋巴細(xì)胞亞群分布的影響來評價(jià)干酪乳桿菌對乳腺癌大鼠免疫功能的調(diào)節(jié)。通過觀察干酪乳桿菌對二甲基苯蒽誘發(fā)乳腺癌大鼠的腸道菌群和腸道連接蛋白表達(dá)的影響來評價(jià)干酪乳桿菌對乳腺癌大鼠腸道屏障功能的調(diào)節(jié)。為進(jìn)一步闡述干酪乳桿菌對二甲基苯蒽誘發(fā)的大鼠乳腺腫瘤的抑制作用機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:1.干酪乳桿菌對二甲基苯蒽誘發(fā)乳腺腫瘤的抑制作用及其免疫功能的影響。(1)動(dòng)物分組及乳腺癌模型建立:60只雌性SD大鼠適應(yīng)性喂養(yǎng)一周后,按體質(zhì)量隨機(jī)分為正常對照組、模型組、干酪乳桿菌劑量1組和干酪乳桿菌劑量2組。模型組及干酪乳桿菌劑量1組和2組大鼠右側(cè)臀部皮下一次性注射100 mg/kg DMBA建立乳腺癌模型。干酪乳桿菌劑量1組和2組分別灌胃給予4 m L/(kg·d)和8 m L/(kg·d)干酪乳桿菌(1×108 CFU/m L),正常對照組和模型組均給予5 m L/(kg·d)大豆油灌胃。每天1次,持續(xù)16周后處死大鼠,腹主動(dòng)脈取血并留取胸腺,脾臟,小腸組織。(2)計(jì)算各組大鼠乳腺癌發(fā)生率、潛伏期、平均瘤重、抑瘤率及臟器指數(shù)。(3)流式細(xì)胞術(shù)檢測大鼠外周血中NK細(xì)胞活性和T淋巴細(xì)胞亞群分布。(4)酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測血清中細(xì)胞因子白介素-4(IL-4)、IL-6、IL-10、IL-12、干擾素-γ(IFN-γ)和腫瘤壞死因子-α(TNF-α)的水平。2.干酪乳桿菌對二甲基苯蒽誘導(dǎo)乳腺癌大鼠腸道菌群和腸道屏障功能的影響。(1)實(shí)時(shí)熒光定量PCR(q PCR)檢測大鼠腸道菌群的變化。(2)HE染色觀察大鼠空腸組織病理學(xué)改變。(3)ELISA檢測大鼠血清中D-乳酸(D-LA)水平。(4)Western Blotting檢測大鼠空腸組織緊密連接蛋白o(hù)ccludin和ZO-1的表達(dá)水平。結(jié)果:1.干酪乳桿菌對二甲基苯蒽誘發(fā)乳腺腫瘤的抑制作用及其免疫功能的影響。(1)正常對照組大鼠無腫瘤發(fā)生,而模型組、干酪乳桿菌劑量1組和2組均有腫瘤發(fā)生。與模型組比較,干酪乳桿菌劑量2組大鼠腫瘤潛伏期延長,腫瘤發(fā)生率和平均瘤質(zhì)量降低(P0.05);抑瘤率達(dá)到41.2%;且該組胸腺指數(shù)顯著升高(P0.05);(2)流式細(xì)胞術(shù)結(jié)果顯示,與模型組比較,干酪乳桿菌劑量2組大鼠TCRαβ+CD161a+NK細(xì)胞百分比、CD3+CD8+T細(xì)胞百分比均顯著升高(P0.05);而血中CD3+Foxp3+細(xì)胞百分比明顯下降(P0.05)。(3)與正常對照組比較,模型組IL-4水平明顯降低,而IL-6、IL-12和TNF-α水平顯著升高(P0.05);與模型組比較,干酪乳桿菌劑量2組血清IL-4、IL-10濃度顯著增高,IL-6、IL-12濃度顯著下降(P0.05);干酪乳桿菌劑量1組和2組TNF-α濃度均顯著下降(P0.05)。2.干酪乳桿菌對二甲基苯蒽誘導(dǎo)乳腺癌大鼠腸道菌群和腸道屏障功能的影響。(1)q PCR結(jié)果顯示,與正常對照組比較,模型組雙歧桿菌、乳酸桿菌和糞腸球菌數(shù)量均下降,其中雙歧桿菌和糞腸球菌數(shù)量下降有統(tǒng)計(jì)學(xué)意義(P0.05);干酪乳桿菌劑量1組和2組雙歧桿菌數(shù)量均比模型組顯著升高(P0.05),而大腸桿菌的數(shù)量有所降低。(2)HE染色結(jié)果顯示,與模型組比較,干酪乳桿菌劑量1組和2組大鼠空腸內(nèi)絨毛和隱窩的形態(tài)明顯完整,結(jié)構(gòu)清晰,絨毛的損傷程度較小。但與正常組比較,干酪乳桿菌劑量1組和2組小腸黏膜厚度減少,絨毛的排列也較為凌亂,絨毛尖端有水腫的現(xiàn)象。(3)ELISA結(jié)果顯示,與正常對照組比較,模型組D-LA濃度顯著升高,而與模型組比較,干酪乳桿菌劑量2組D-LA濃度顯著下降(P0.05)。(4)Western Blotting結(jié)果顯示,與正常對照組比較,模型組緊密連接蛋白o(hù)ccludin和ZO-1的表達(dá)均顯著下降,而干酪乳桿菌劑量2組均比模型組顯著升高(P0.05)。結(jié)論:1.干酪乳桿菌對大鼠乳腺腫瘤的生長具有一定的抑制作用。2.干酪乳桿菌可調(diào)節(jié)CD4+、CD8+T細(xì)胞、NK細(xì)胞及調(diào)節(jié)性T細(xì)胞等免疫細(xì)胞分布,改善炎性相關(guān)細(xì)胞因子水平,提高機(jī)體免疫調(diào)節(jié)作用。3.干酪乳桿菌可改善腸道菌群的分布,降低血清中D-LA濃度、增加腸道緊密連接蛋白o(hù)ccludin、ZO-1的表達(dá),提高機(jī)體的腸道屏障功能。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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