【作者】 托娅；

【导师】 张和平；

【作者基本信息】 内蒙古农业大学 ， 畜产品安全生产， 2008， 博士

【摘要】 本课题以内蒙古农业大学乳品生物技术与工程教育部重点实验室从传统发酵酸马奶中分离鉴定的240株乳杆菌中,经前期大量实验确立的一株具有明显益生特性的乳杆菌Lactobacillus. casei Zhang(Lb. casei Zhang)为研究对象,观察了其对实验小鼠的的免疫调节及抗肿瘤作用,并探讨了其可能的作用机理。前期的大量研究表明,益生菌Lb. casei Zhang具有优良的益生特性和降胆固醇、抗氧化和免疫调节等对机体有益的功能特性。本课题以前期的研究为基础,深入探讨了益生菌Lb. casei Zhang免疫调节及抗肿瘤方面的功能特性,为其进一步开发应用提供了实验依据。本课题实验分四部分:第一部分:采用动物活体试验方法,Lb. casei Zhang制备成热致死菌体和活菌制剂各分成高、中、低三个剂量组灌服小鼠15天,测定胸腺指数、脾脏指数;观察ConA促进脾淋巴细胞增殖作用、脾脏巨噬细胞吞噬中性红的能力。采用ELISA法检测血清中IFN-γ、IL-12、IL-1α、TNF-α的含量。第二部分: Lb. casei Zhang活菌制剂分高、中、低三个剂量组灌服小鼠。分别于第3、7、11、15天,摘取脾脏,计算脾指数;RT-PCR法测定脾脏中IL-2和IL-2RαmRNA的转录水平,并从小鼠局部肠粘膜免疫组织及免疫细胞着手,对服用Lb. casei Zhang后小鼠肠道局部免疫组织一Peyer’s结的数量计分进行了研究。采用免疫组化法检测小肠粘膜CD3+、IgA+细胞数量并通过图象分析软件分析其阳性信号平均光密度值了解其表达情况。第三部分:以荷H22肝癌小鼠为模型,用Lb. casei Zhang活菌制剂分高、中、低三个剂量灌服小鼠12天后,滴片法测定小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数;计算脾指数,计算肿瘤生长抑制率,ELISA法测定血清中肿瘤坏死因子-β(TNF-β)的含量;免疫组化法检测小鼠肿瘤细胞中癌基因Bcl-2和抑癌基因Bax蛋白的表达程度,从分子水平探讨其抗肿瘤的可能机制。第四部分:以荷H22肝癌化疗小鼠为模型,观察Lb. casei Zhang对化疗药物的减毒增效作用。以腹腔注射环磷酰胺(CTX)联用Lb. casei Zhang活菌制剂高、中、低三个剂量灌服小鼠12天后,计算脾指数、肿瘤生长抑制率,并对小鼠肠道局部免疫组织一Peyer’s结进行计数计分;采用原位杂交法检测肿瘤细胞中增殖细胞核抗原(PCNA)的表达,采用碘化丙啶(PI)染色利用流式细胞仪检测肿瘤细胞细胞周期及凋亡率。通过以上的实验结果,我们得出的总体结论是:具有明显益生特性的乳杆菌Lb. casei Zhang对实验小鼠的细胞免疫、体液免疫、肠道粘膜免疫可产生多方面的、有利于机体免疫功能状态的调节作用;对体外移植性小鼠肝癌H22肿瘤具有明显的抑制生长作用,对化疗药环磷酰胺(CTX)具有明显的减毒增效作用,其抗肿瘤的机制可能是通过增强荷瘤小鼠的免疫功能、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡而发挥作用。更多还原

【Abstract】 On the basis of the research on the 240 strains of Lactobacillus. casei Zhang(Lb. casei Zhang)isolated from the traditionally home-made koumiss by the Key Laboratory of Dairy Biotechnology and Engineering of Inner Mongolia Agricultural University affiliated to the Ministry of Education. This thesis studies the function of immunity adjustment and anti-tumor activity of one strain of probiotic bacterium Lb. casei Zhang in the body of a laboratory mouse, which contains the potential probiotic properties proved in many past experiments, and explores its possible mechanism of activity.According to the rich studies in the early stage, it is proved that the Lb. casei Zhang has good probiotic properties, and some functions such as reducing cholesterin, antioxidation, and immune adjustment as well as other properties.Based on the studies in the early stage, this thesis further explores the immunity adjustment and anti-tumor activity of Lb. casei Zhang, and then lays a foundation of research in experiment for its further development and application.The experiment on this thesis consists of four parts:Part I:Taking the in vivo experiment, several little mice are orally taken the bacillus killed by high heat and live bacillus made from Lb. casei Zhang in three dosages with high dose, middle dose and low dose and observed for 15 days. Measure the thymus index and spleen index of the mice; observe the function and ability of ConA in the lymphocyte multiplication and macrophage phagocytic function. Test the content of IFN-γ, IL-12, IL-1αand TNF-αin the blood serum in the method of ELISA.Part II:Live bacillus of Lb. casei Zhang is divided into three dosage of high, middle and low and instilled into the mice. And then remove their spleens to calculate the index of spleens at the third, seventh, eleventh and fifteenth day. Take the RT-PCR method to measure the transcription level of IL-2 and IL-2RαmRNA in the spleens.Set about the research on the immune tissue and immune cells of local intestinal mucosa of little mice, study the number counting of Peyer’s in the local intestine immune tissue of mice taken Lb. casei Zhang, measure the number of CD3+ and IgA+ of small intestine mucous by the method of immune histochemistry, and then analyze the average optical density with positive signal by using image analysis system so as to know its expressive condition.Part III:Take the little mice with H22 liver cancer for example, 12 days later when the mice are instilled into live bacillus of Lb. casei Zhang in three dosages of high, middle and low, and measure the rate and index of peritoneal macrophage phagocytizing chicken red blood cell in mice by drop technique and calculate the index of spleen and the rate of tumor suppression. Measure the number of Tumor Necrosis Factorβin blood serum by the method of ELISA, check the expression of cancer bcl-2 gene and tumor suppressor gene by immune histochemistry methods, and then explore the possible mechanism of tumor suppressor based on the molecule level.Part IV:Take the mice with H22 liver cancer treated by chemical therapy as models, and then observe the action of toxin-attenuating and efficacy– enhancing caused by Lb. casei Zhang. 12 days later when the mice are injected with Cyclophosphamide in the abdomen and together instilled into live bacillus of Lb. casei Zhang in three dosages of high, middle and low, calculate the index of spleen and rate of tumor suppression, record the Peyer’s in the local immune tissue of mice’s intestine, check the expression of PCNA in tumor cells by using the method of in situ hybridization, and check the cell cycle and apoptosis rate of tumor cells by using flow cytometer with propidium iodide technology.We reach a general conclusion based on the above experiments: the Lb. casei Zhang with evident probiotic properties have many active adjustment actions on cell immunity, humoral immunity and intestine mucosal immunity of laboratory little mice; have an evident inhibitory action of growth on the transplanted tumor of H22 liver cancer-bearing mice; have an evident action of toxin-attenuating and efficacy– enhancing on the Cyclophosphamide in chemical therapy. Its mechanism to resist tumor probably is to inhibit the increase of tumor cells and induce the apoptosis of tumor cells through strengthening the immunity of mice with tumor.更多还原