草分枝杆菌制品对实验小鼠免疫功能的影响研究

发布时间：2018-03-05 15:29

  本文选题：草分枝杆菌　切入点：小鼠　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：草分枝杆菌(Mycobacterium phlei)是一种在自然界广泛存在的天然药物资源,含有多种免疫活性成分。在医学领域中,草分枝杆菌注射剂乌体林斯已经被广泛应用于紫癜、哮喘等多种疾病的治疗,但是其应用于免疫方面的研究很少,特别是在畜牧业生产中,草分枝杆菌应用的报道非常罕见,同时未见有比较不同处理方法制备的草分枝杆菌对动物免疫功能影响的研究报道。为此,本研究主要探讨不同方法获得的草分枝杆菌制品对小鼠免疫功能的影响。首先随机选取10只小鼠进行急性毒性试验,小鼠灌服草分枝杆菌菌液0.2mL/天/只,其中含有草分枝杆菌有效成分110mg。然后选取120只体重18-22g的昆明小鼠,随机分为5组,试验组分别为草分枝杆菌活菌组、细胞壁组、热灭活组、超声破碎组,以生理盐水为对照。每组24只,4个草分枝杆菌试验组分别灌服不同方法获得的草分枝杆菌制品,剂量为110mg/只/d,灌胃体积为0.2mL,定期称量小鼠体重,6周后每组随机选取6只小鼠,测定其胸腺指数和脾脏指数,用抗凝血测定其血常规及部分肝肾功能指标;然后每个试验组再取6只小鼠取血,分离血清用比浊法测定血清中溶菌酶,用生物素双抗体夹心酶联免疫吸附法测定血清IL-2和IgG含量;无菌分离脾脏并制备脾细胞悬液用MTT法测定其淋巴细胞转化率和NK细胞活性;再每组另取6只小鼠,测定其腹腔巨噬细胞吞噬酵母菌能力;每组最后剩余的6只小鼠攻鼠伤寒沙门菌,进行免疫保护试验。结果表明:①急性毒性试验结果显示:草分枝杆菌对试验小鼠无毒。②5组小鼠血常规指标PLT、MPV、PDW和P-LCR以及肝肾功能指标ALB、TCHE、UREA、CRE和TG水平均差异不显著;而对于血常规中的WBC、RBC、HGB以及HCT等指标和肝肾功能指标中的ALP、TP以及ALT等指标的水平,草分枝杆菌4个试验组均高于生理盐水组,但是均在正常范围内。③草分枝杆菌4个试验组对小鼠体重的影响与对照组相比无显著性差异。④对于小鼠的脏器指数,草分枝杆菌4个试验组的脾脏指数和胸腺指数均显著高于生理盐水组,其中细胞壁组的脾脏指数显著高于其余3组,但后三者之间差异不显著;活菌组和细胞壁组的胸腺指数显著高于热灭活组和超声破碎组,而后两者差异不显著。⑤草分枝杆菌4个试验组的淋巴细胞转化率、NK细胞活性、巨噬细胞吞噬功能和血清IL-2含量均显著高于生理盐水组;其中活菌组和细胞壁组的刺激指数差异无统计学意义,但显著高于热灭活组和超声破碎组,而后二者差异不显著;细胞壁组和热灭活组的NK细胞活性和IL-2含量差异不显著,但较活菌组和超声破碎组明显提高,后二者之间差异无统计学意义;而对于草分枝杆菌4个试验组的巨噬细胞吞噬功能,细胞壁组显著高于其余三组,活菌组和热灭活组之间无显著性差异,但显著高于超声破碎组。⑥草分枝杆菌4个试验组的血清中溶菌酶的活性均显著高于生理盐水组,热灭活组的溶菌酶活性显著高于活菌组、细胞壁组和超声破碎组,但后三者之间差异不显著;草分枝杆菌细胞壁组、热灭活组的血清IgG含量显著高于活菌组、超声破碎组以及生理盐水组,但后三者之间差异无统计学意义。○7灌胃结束后,通过注射沙门菌进行小鼠攻毒试验,攻毒后7天,免疫保护试验结果显示,草分枝杆菌活菌组、细胞壁组、热灭活组、超声破碎组和生理盐水组对小鼠的攻毒保护率分别为33.3%、66.7%、66.7%、50%和0,表明草分枝杆菌能在一定程度上增强小鼠的细胞免疫和体液免疫功能。综上说明,草分枝杆菌是一种安全有效的免疫调节剂,能明显增强小鼠的免疫能力。
[Abstract]:Mycobacteriumphlei (Mycobacterium phlei) is a kind of widely existed in the nature of natural medicine resources, containing a variety of immune activity components. In the field of medicine, mycobacteriumphlei Utilins injection has been widely used in the treatment of purpura, asthma and other diseases, but is rarely used in the study of the immunity, especially in animal husbandry in addition, mycobacteriumphlei application reports are very rare, and there has been no report about the comparison of different treatment methods of Mycobacterium phlei preparation effect on animal immune function. Therefore, this study aims to investigate the effect of Mycobacterium phlei products obtained by different methods on immune function of mice. The first 10 mice were randomly selected for acute toxicity test, mice fed mycobacteriumphlei bacteria 0.2mL/ / day, which contains the effective components of Mycobacterium phlei 110mg. then selected 120 weight 18-22g Kunming The mice were randomly divided into 5 groups, experimental group were mycobacteriumphlei bacteria group, cell wall components, heat inactivated group, ultrasonic group, physiological saline control group. Each group had 24 rats, 4 mycobacteriumphlei experimental groups were obtained by taking different methods of mycobacteriumphlei products, dose 110mg/ /d, intragastric volume was 0.2mL, the weight of mice after 6 weeks of regular weighing, each group of 6 mice were randomly selected, the determination of the thymus index and spleen index, the determination of blood with anticoagulant and part of liver and kidney function index; then each experimental group and 6 mice were separated from blood serum by determination of serum lysozyme turbidimetric method, determination of serum IL-2 and IgG content with biotin double antibody sandwich enzyme-linked immunosorbent assay; sterile isolated spleen and preparation of spleen cell determination of conversion rate and NK cell activity in the cell suspension with MTT method; then another 6 mice in each group, the determination of peritoneal macrophages Cell phagocytosis of yeast; 6 mice in each of the last remaining Salmonella typhimurium, immune protection test. The results showed that: the results of acute toxicity test showed that Mycobacterium phlei mice non-toxic. In the 5 groups of Mice Blood Routine Indexes of PLT, MPV, PDW and P-LCR as well as liver and kidney function indexes of ALB, TCHE, UREA. CRE and TG were no significant difference in blood; and for WBC, RBC, HGB, HCT and other indicators of liver and kidney function indexes of ALP, TP, ALT and other indicators of the level, mycobacteriumphlei 4 test groups were higher than that of saline group, but were within the normal range. 3 mycobacteriumphlei 4 a group of test of influence on body weight of mice compared with the control group had no significant difference. 4 for the organ index of mice, mycobacteriumphlei 4 in experimental group, spleen index and thymus index were significantly higher than those in the saline group, the cell wall group spleen index significantly Compared with the other 3 groups, but no significant difference between the three groups; the thymus index of live bacteria and cell wall group was significantly higher than that of heat inactivated group and ultrasonic group, and there was no significant difference between them. 5 mycobacteriumphlei 4 in experimental group, lymphocyte transformation rate, NK cell activity, the function and the content of serum IL-2 phagocytosis of macrophages were significantly higher than those in the saline group; there was no significant bacterial cell wall group group and the stimulation index difference, but was significantly higher than that of heat inactivated group and ultrasonic group, and the difference between the two is not significant; the difference of NK cell activity and IL-2 content of cell wall components and heat inactivated group is not obviously, but compared with live bacteria group and ultrasonic group increased significantly, there was no significant difference between the two; and for mycobacteriumphlei 4 test group of macrophage phagocytosis, cell wall group was significantly higher than the other three groups, between live bacteria group and heat inactivated group A significant difference, but significantly higher than ultrasonic group. The serum lysozyme mycobacteriumphlei 4 test groups in the activity were significantly higher than those in the saline group, heat inactivated group of lysozyme activity was significantly higher than that of live bacteria, cell wall group and ultrasonic group, but no significant difference between the latter three Mycobacterium; coli cell wall components, heat inactivated serum content of IgG group was significantly higher than that of live bacteria group, ultrasonication group and normal saline group, but there was no significant difference between the three. 7 hours after gavage, mice were challenged by Salmonella test injection, 7 days after infection, the immune protection test results Mycobacterium phlei, living bacteria group, cell wall components, heat inactivated group, ultrasonic group and saline group of mice infected protection rates were 33.3%, 66.7%, 66.7%, 50% and 0, showed that Mycobacterium phlei can enhance mouse cell immunity and body to a certain extent In conclusion, Mycobacterium Mycobacterium is a safe and effective immunomodulator, which can obviously enhance the immune ability of mice.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79
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本文编号：1570823