维生素D缺乏动物模型的建立及其应对结核抗原的免疫变化

发布时间：2018-01-09 07:38

本文关键词：维生素D缺乏动物模型的建立及其应对结核抗原的免疫变化　出处：《第三军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景及目的: 中国是结核高疫情国家,是世界上22个结核病高负担国家之一,并且为耐药结核病疫情最严重的国家之一[1]。第四次全国结核病流行病学抽样调查显示全国结核发病率平均为367/10万人,而重庆地区为549/10万人,是结核病的高发地区,每年新发现4万结核病患者,是京津沪的10余倍,其原因目前尚不清楚。重庆是著名的山城,地理气候比较独特,其年日照量全国最低,紫外线照射严重不足。前期调查发现重庆地区63.16%的健康献血者与66.67%的骨关节结核患者存在维生素D缺乏。VD对免疫细胞的功能有着多方面的作用,体外研究表明VD既可增强巨噬细胞的吞噬能力,又可抑制DC细胞成熟,并影响T细胞的分化及功能[2]。 (一)1 ,25 (OH) 2 D3及其衍生物具有增强天然免疫作用,研究表明VD缺乏与结核易感性密切相关,随着25(OH)D水平的升高,感染危险降低,潜伏感染发展为结核病的危险也降低[3]。在结核化疗前时代,光照疗法和营养疗法是治疗结核病的主要措施,经皮肤合成和饮食摄入增加了体内25 (OH) D和1 ,25 (OH) 2 D3水平,结核治愈率提高至约25%,抗结核药物发明后VD在结核病治疗中逐渐被淡忘。近年来,VD免疫调节作用研究的兴起、结核耐药与抗结核药物研发滞后的矛盾促使学者们重新重视VD在防治结核病中的应用[4]。重庆地区独特的地理和气候特点导致年日照量全国最低,紫外线照射不足而皮肤合成的VD较少,因贫困经饮食摄取VD不足,冬季VD缺乏较为普遍。VD与免疫系统的关系近年来受到广泛关注,通过黄光照射、去VD合成营养素饲料喂养建立的小鼠VD缺乏模型,免疫机制严重紊乱,表现为T细胞亚群分布失调、免疫耐受减弱而自体免疫损伤加重,T淋巴细胞亚群分布紊乱、功能下降(Th2抑制),提示1 ,25 (OH) 2 D3在单核/巨噬细胞分化成熟中的重要意义,补充VD 8周后可恢复正常;因建立条件严格、耗时长,佝偻病研究中未被广泛采用,但该模型为研究环境因素导致VD缺乏对免疫系统影响的最理想模型。 (二)1 ,25 (OH) 2 D3及其衍生物具有诱导免疫耐受减轻免疫自体损伤作用。1 ,25 (OH) 2 D3能够影响DCs生命周期中所有的主要阶段:阻碍单核细胞分化成DCs;阻止幼稚DCs向成熟DCs分化, MHC-Ⅱ类分子和共刺激分子CD40、CD80、CD86表达下调,IL-12分泌减少,IL-10显著增加,从而诱导出具有致耐受性表型和功能的未成熟DCs,未成熟DCs与T淋巴细胞的耐受性相关。然而VD缺乏是否是结核的易感因素尚需要体内实验进一步证明。为研究VD缺乏在结核感染发病中的作用,我们首先建立了VD缺乏小鼠模型,然后用卡介菌刺激,观察体内的免疫变化与正常小鼠刺激后的差别。方法: 1.维生素缺乏纯合成小鼠饲料的配制将双蒸水50L平均分装到容量为50L的金属桶装容器中,分别加入625g琼脂,在105℃加热40分钟,然后放入到60℃的保温箱中备用。将比例[3]称量的各种原料放入混合机中混合30min,后加入到无尘粉碎机中粉碎,全部一次性通过200钼筛。将其平均分成两份分别加入到琼脂溶液中,并加入脂溶性维生素溶液各250ml(一份含VD,一份未含),充分搅拌30min,室温下冷却1h,然后放入到-20℃冰箱中冻存。 2.维生素缺乏动物模型的建立及评价 36只怀孕2周的BALB/C小鼠,分为三组。正常组喂正常饲料、VD+组喂VD+饲料、VD-组喂VD-饲料。VD-组遮光黄光灯照射。剔除雄性子鼠,入组的子鼠正常组18只、VD+组15只、VD-组13只,出生3周断奶,饲养环境及饮食同母鼠。测4-10周、12周子鼠体重,8、12周时测每天子鼠饮食消耗量,同时观察子鼠运动习性。12周时将上述三组子鼠剪尾取血100ul离心取血清测VD水平,每组随机取三只子鼠摘眼球取血,测白蛋白水平;将VD缺乏子鼠10只随机分为组I继续遮光+正常饮食,组II正常环境+正常饮食,6周后摘眼球取血测VD水平。 3.VD缺乏子鼠体内免疫机能变化的检测尾静脉取血测VD后将上述三组子鼠每组取5只分别在其尾根部注射BCG(0.1mlBCG含卡介苗0.05mg)0.1ml,再饲养六周,饲养环境同前。刺激6周后分离血清,-70℃冻存待检;无菌分离脾淋巴细胞,调整浓度为5×10~6 cell/ml。取200μl脾淋巴细胞悬液,流式细胞仪检测CD4 +和CD8 +T细胞百分比。间接ELISA方法测定特异性IFN-γ和IL-10水平,及血清特异性抗体滴度。CCK-8检测脾淋巴细胞增殖情况,结果用刺激指数(Stimulation index,SI)表示,SI=A实验孔/A对照孔。如刺激指数1,则用Annexin V-PI试剂盒检测是否凋亡。结果: 1.正常组与VD+组体重无统计学差异(P0.05);VD+组、VD-组8周、12周日平均消耗饲料,经统计学分析8周时有差异,12周时无差异。 2.正常组、VD+组、VD-组血浆白蛋白分别为33.2±1.04、32.4±0.53、32.8±0.67(g/L),无统计学差异。 3.正常组与VD+组血浆VD水平无显著差异,但VD-组血浆VD含量下降到正常的20%以下,提示小鼠VD缺乏模型建立成功;六周后,组I、组II VD水平分别为18.46±1.53、58.46±5.53(nmol/L)(P0.001)。 4.测子鼠血浆钙水平分别为2.32±0.087、2.49±0.144、1.73±0.091(mmol/L),血浆磷水平分别为2.20±0.071、2.41±0.042、1.77±0.091(mmol/L),经统计学分析三组之间血浆钙磷含量均有统计学差异(P0.05),正常组与VD+组无显著差异(P0.05),VD-组与另外两组均有显著差异(P0.05)。 5.BCG刺激正常组、VD+组及VD-组6周后,脾淋巴细胞中CD4+细胞亚群百分率分别为28.64±0.59、24.86±0.49、30.1±0.8, CD8+为10.9±0.72、9.42±0.55、14.14±0.83, CD4+/CD8+的比值分别为2.64±0.19、2.66±0.23、2.13±0.09,VD-组CD4+及CD8+淋巴细胞百分比均显著上升尤其以CD8+上升幅度更大。 6.血浆IFN-γ分别为375.40±13.11、301.96±23.60、478.43±17.50(pg/ml),血浆IL-10分别为27.35±2.81、28.55±0.72、21.47±1.34(pg/ml),VD-组较其他两组血浆IFN-γ显著上调而IL-10显著下调。 7.VD-组血清特异性抗TB-PPD抗体显著高于正常组及VD+组。 8.三组脾淋巴细胞增值刺激指数分别为1.47±0.12、1.15±0.11、0.65±0.16,VD-抑制了脾脏T淋巴细胞在TB-PPD刺激下的增殖能力,其中VD-组淋巴细胞出现显著凋亡。结论: 1.通过控制食物和光照,成功建立VD缺乏BALB/c小鼠动物模型。 2.在VD的天然来源中,紫外线的作用较饮食作用重要,但直接补充VD效果显著。 3.VD缺乏可引起一系列疾病,在本实验中出现佝偻病症状。 4.VD缺乏显著改变了机体免疫系统对BCG刺激的应答特征,提示可能与VD缺乏容易引起结核病相关。 5.VD在天然免疫中发挥重要病因学作用,同时增加免疫耐受,减轻自身免疫性损伤。这为临床治疗结核感染及自身免疫性疾病提供一条崭新的思路。
[Abstract]:Background and purpose:
China is high tuberculosis epidemic state, is one of the world's 22 TB high burden countries, and is one of the most serious drug-resistant TB epidemic in the country [1]. fourth national epidemiological sampling survey of tuberculosis showed the incidence of TB was 367/10 million, while the Chongqing area 549/10 million people, is a high incidence of tuberculosis, 40 thousand were new TB patients each year, Beijing Tianjin and Shanghai is more than 10 times, the reason is unclear. Chongqing is the famous mountain, unique geography and climate, the annual sunshine with the lowest amount of ultraviolet radiation is seriously insufficient. The investigation found that Chongqing area of 63.16% healthy blood donors in patients with osteoarticular tuberculosis and 66.67% in the presence of vitamin D a number of roles of.VD lack of immune cell function in vitro studies showed that VD can enhance the phagocytosis of macrophage, but also inhibit the maturation of DC cells, and the influence of T fine Cell differentiation and function [2].
(a) 1, 25 (OH) 2 D3 and its derivatives with enhanced natural immunity, lack of research shows that VD is closely related with the susceptibility to tuberculosis, 25 (OH) with elevated levels of D, reduce the risk of infection, latent infection development risk of TB also decreased [3]. in tuberculosis chemotherapy era, light therapy and nutrition therapy is the main measures for treatment of tuberculosis, the skin synthesis and dietary intake increased in 25 and 1 D (OH), 25 (OH) 2 D3, tuberculosis cure rate increased to about 25%, anti tuberculosis drugs invented after VD in the treatment of tuberculosis was gradually forgotten. In recent years, the rise of VD immunoregulation the contradiction between tuberculosis and anti TB drug research lag makes scholars attach great importance to the unique geographical and climate characteristics in the application of [4]. VD in prevention and treatment of tuberculosis in Chongqing area resulted in the lowest amount of annual sunshine and ultraviolet irradiation skin synthesis of V D less, due to poor dietary intake by VD is insufficient, lack of common relationship between winter VD.VD and immune system attracted widespread attention in recent years. Through the yellow light irradiation to mouse VD VD synthetic diet to establish the lack of model, serious disorder of immune mechanism for T cell subsets disorders, immune tolerance weakened and autoimmune injury, T lymphocyte subsets distribution disorder, functional decline (Th2 inhibitor), suggesting that the 1, 25 (OH) 2 important D3 in monocyte / macrophage differentiation, VD 8 weeks after the return to normal; because of the establishment of strict conditions, time-consuming, rickets is not on widely used, but the model for the study of environmental factors cause the ideal model of VD lack of effect on the immune system.
(two) 1, 25 (OH) 2 D3 and its derivatives with immune tolerance induced by autologous immune reduce the damage effect of.1, 25 (OH) 2 D3 to all the main stages in the life cycle of DCs block the differentiation of monocytes into DCs; prevent the differentiation of naive DCs DCs, MHC- class II and costimulatory molecules CD40, CD80, CD86 expression, IL-12 secretion decreased, IL-10 increased significantly, which is caused by DCs induced immature phenotype and function of tolerance, tolerance of immature DCs and T lymphocytes.
However, the lack of VD is a susceptibility factor for tuberculosis in vivo still need further proof. For the study of VD deficiency in the pathogenesis of tuberculosis infection, we first establish a VD deficient mouse model, and then stimulated with BCG immune changes observed in vivo and in normal mice after stimulation.
Method:
Preparation of 1. vitamin lack of pure synthetic mice feed
The double distilled water 50L the average capacity of metal packaging to bottled container 50L, were added to the 625g agar, heated at 105 DEG C for 40 minutes, then add to the spare box 60 degrees. The proportion of raw materials weighing [3] into mixing 30min, adding to clean and crushing, through all the time 200 Mo sieve. The average is divided into two parts were added to the agar solution, and add fat soluble vitamin 250ml solution (including a VD, a not included), fully mixing 30min, 1H cooling at room temperature, and then put into the refrigerator to -20 DEG C and frozen.
Establishment and evaluation of an animal model of 2. vitamin deficiency
36 only 2 weeks pregnant BALB/C mice were divided into three groups. The control group fed with normal diet, group VD+ fed VD+ diet, VD- group fed VD- diet group.VD- shading yellow light irradiation. Excluding male mice, the group were 18 rats in the normal group, 15 rats in group VD+, 13 rats in group VD-, born 3 week of weaning, feeding environment and diet were measured. The same 4-10 weeks, weighing 12 weeks in sub 8,12 weeks were measured every day, food consumption, and exercise characteristic was observed at.12 weeks of the three group were cut the tail blood serum were measured 100ul VD levels of each group were randomly selected three offspring mice eyeballs. Blood test, albumin level; VD lack of offspring 10 were randomly divided into group I to group II shading + normal diet, normal environment + normal diet, 6 weeks after the eyeball blood VD level.
3.VD lack of detection of changes in immune function in rats
The tail vein blood were measured after VD of the three group were 5 rats in each group were only in the root of the tail injection of BCG (0.1mlBCG 0.05mg 0.1ml, including BCG) and then fed for six weeks. Before stimulation feeding environment 6 weeks after separation of serum, -70 C frozen for inspection; aseptic splenic lymphocytes, adjust the concentration of 5 * 10~6 cell/ml. 200 L spleen lymphocyte suspension, flow cytometry and CD8 CD4 + +T cell percentage. Determination of specific IFN- and IL-10 levels of indirect ELISA method, and serum specific antibody titer.CCK-8 detection of splenic lymphocyte proliferation, the stimulation index (Stimulation index SI) said, SI=A experimental hole /A control holes. Such as the stimulation index of 1, using the Annexin V-PI kit to detect whether apoptosis.
Result:
1., there was no significant difference in body weight between normal group and VD+ group (P0.05). In group VD+, group VD- consumed 8 weeks, 12 weeks on average, and consumed 8 times a week. There was no difference between 8 weeks and 12 weeks.
The plasma albumin of 2. normal group, VD+ group and VD- group was 33.2 + 1.04,32.4 + 0.53,32.8 + 0.67 (g/L), and there was no statistical difference.
3. there was no significant difference in plasma VD level between normal group and VD+ group, but plasma VD content in group VD- dropped to less than 20% of normal level, suggesting that the VD deficient model of mice was established successfully. After six weeks, the II VD level of group I was 18.46 + 1.53,58.46 + 5.53 (nmol/L) (P0.001).
4. measurements of plasma calcium levels were respectively 2.32 + 0.087,2.49 + 0.144,1.73 + 0.091 (mmol/L), serum phosphorus levels were 2.20 + 0.071,2.41 + 0.042,1.77 + 0.091 (mmol/L), there was statistical plasma calcium and phosphorus content difference between the three groups (P0.05), there was no significant difference between the normal group and VD+ group (P0.05). The VD- group and the other two groups had significant difference (P0.05).
The stimulation of 5.BCG normal group, VD+ group and VD- group after 6 weeks, spleen lymphocyte CD4+ cell subsets was 28.64 + 0.59,24.86 + 0.49,30.1 + 0.8, CD8+ = 10.9 + 0.72,9.42 + 0.55,14.14 + 0.83, CD4+/CD8+ ratio were 2.64 + 0.19,2.66 + 0.23,2.13 + 0.09, VD- group CD4+ and CD8+ lymphocyte percentage were significantly the CD8+ Rose Rose more steeply.
6., plasma IFN- gamma was 375.40 + 13.11301.96 + 23.60478.43 + 17.50 (pg/ml), plasma IL-10 was 27.35 + 2.81,28.55 + 0.72,21.47 + 1.34 (pg/ml), VD- group was significantly higher than other two groups, and IL-10 significantly decreased.
The serum specific anti TB-PPD antibody in 7.VD- group was significantly higher than that of the normal group and the VD+ group.
8., the three groups of spleen lymphocyte proliferation stimulation index were 1.47 + 0.12,1.15 + 0.11,0.65 + 0.16, VD- inhibited the proliferation of spleen T lymphocytes stimulated by TB-PPD, and VD- group had significant apoptosis.
Conclusion:
1. the animal model of VD deficient BALB/c mice was successfully established by controlling the food and light.
2. in the natural source of VD, the effect of ultraviolet light is more important than diet, but the effect of direct supplement of VD is significant.
The deficiency of 3.VD can cause a series of diseases and the symptoms of rickets in this experiment.
4.VD deficiency significantly changes the response characteristics of the immune system to BCG stimulation, suggesting that it may be associated with the lack of VD to cause tuberculosis.
5.VD plays an important role in the etiology of innate immunity, and at the same time, increases immune tolerance and reduces autoimmune injury. This provides a new way for clinical treatment of tuberculosis infection and autoimmune diseases.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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