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BACKGROUND AND PURPOSE: China is a the high tuberculosis epidemic countries, is one of the 22 high TB ??burden countries in the world, and is one of the most serious outbreaks of drug-resistant TB countries [1]. Fourth National TB epidemiological survey shows the national average incidence of TB is 367/10 million, and the Chongqing region of 549/10 million people, is the high incidence of TB region, newly discovered 40,000 TB patients each year, the Beijing-Tianjin Shanghai's more than 10 times, the reason is not clear. Chongqing is a well-known mountain town, unique geography and climate, a serious shortage of national minimum annual amount of sunlight, ultraviolet radiation. The preliminary survey found that 63.16% of healthy blood donors in Chongqing area, 66.67% of patients with bone and joint tuberculosis vitamin D deficiency exists. VD have many of the functions of immune cells, in vitro studies have shown that VD can enhance the phagocytic activity of macrophages, but also inhibit DC maturation and influence the differentiation and function of T cells [2]. (A) 1, 25 (OH) 2 D3 and its derivatives have enhanced natural immunity, studies have shown that VD lack closely related to tuberculosis susceptibility with 25 (OH) D levels increased the risk of infection to reduce latent infection development also reduces the risk of TB [3]. Light therapy and nutritional therapy in tuberculosis before chemotherapy era, the main measures for the treatment of tuberculosis, increased synthesis and dietary intake through the skin in vivo 25 (OH) D and 25 (OH) 2 D3 level, TB cure rate increased to about 25%, VD invention of anti-TB drugs in TB treatment gradually being forgotten. In recent years, the VD immunomodulatory effect of the rise of tuberculosis resistant to anti-TB drug research and development lag contradiction prompted scholars to a renewed emphasis on the VD TB [4]. Chongqing unique geographical and climatic characteristics of the region led to the national minimum amount of annual sunshine, ultraviolet radiation and skin synthesis fewer VD VD inadequate due to poor dietary intake, lack of winter VD is more common. The VD and the relationship of the immune system in recent years, attracted widespread attention through the yellow light exposure to VD the synthetic nutrients fed mice established VD lack model serious disorders of the immune mechanism, the performance of the distribution of T cell subsets disorders, weakening of the immune tolerance and autologous immune injury aggravated, T lymphocyte subsets distribution disorders, functional decline (Th2 inhibition), suggesting that 1, 25 (OH) 2 D3 recoverable in monocyte / macrophage differentiation significance of the mature, supplement VD 8 weeks normal; establish strict conditions, time-consuming, rickets study has not been widely adopted, but the VD model for the study of environmental factors lead to a lack of impact on the immune system the best model. (B) 1, 25 (OH) 2 D3 and its derivatives have a role in the induction of immune tolerance to reduce immune autologous injury. 1, 25 (OH) 2 D3 can affect the the DCs life cycle in all main stages: impede monocyte differentiation into DCs; to prevent immature DCs to mature DCs differentiation, MHC-II molecules and costimulatory molecules of CD40, CD80 and CD86 expression downward reduced secretion, IL-12, IL-10 significantly increased, thereby inducing tolerance related with a tolerogenic phenotype and function of immature DCs, immature DCs and T lymphocytes. However VD deficiency are predisposing factors of TB is still need for in vivo experiments to further prove. To study the of VD lack of role in the pathogenesis of tuberculosis infection, we first establish the VD lack of a mouse model, and then use the BCG to stimulate the observed differences in the changes in the body's immune and normal mice stimulated. Method: 1. Vitamin deficiency mouse pure synthetic feed preparation double distilled water 50L average loaded to capacity of 625g agar 50L of the metal drums were added and heated for 40 minutes at 105 ℃, and then put into 60 ° C incubator spare. Ratio [3] weighing various raw materials into the mixer 30min, after adding to the dust-free pulverizer pulverizing all the time through the sieve of 200 molybdenum. Its average is divided into two separately added to the agar solution and the fat-soluble vitamin solution was added 250ml (a containing VD, a not included), sufficiently stirred 30min, the mixture was cooled at room temperature for 1h, then placed in refrigerator to -20 ℃ in cryopreservation. 36 2 weeks pregnant BALB / C mice of vitamin deficiency establishment and evaluation of animal models, divided into three groups. Normal group was fed a normal diet, the VD group fed VD feed, VD-group was fed the VD-feed. The VD-groups blackout yellow light irradiation. Eliminate male offspring, into the group's sub-rat normal group 18, VD group 15, VD-group of 13, was born three weeks weaning, rearing environment and diet and female rats. Measured 4-10 weeks, 12 weeks offspring body weight measured at 8 and 12 weeks of daily dietary consumption of offspring, while observing the offspring movement habits. 12 weeks, the three groups of sub-rat tail - cut 100ul centrifugal serum was measured VD level, each randomly selected the three sub mice eyeballs to take blood, measured albumin levels; lack the VD 10 offspring were randomly divided into groups I continue shading normal diet, group II normal environment normal diet, six weeks after the blood to determine VD level eyeballs. Detection 3.VD lack of in vivo changes in immune function in offspring tail vein of the three groups will blood to determine VD offspring each group to take 5, respectively, in the base of the tail injection the BCG (0.1mlBCG containing BCG 0.05mg) 0.1 ml of re-feeding six weeks, the same as the breeding environment. The separation of serum stimulation after 6 weeks, -70 ℃ cryopreservation be examined; aseptic isolation of splenic lymphocytes, and adjust the concentration of 5 × 10 6 cell / ml. Take 200μl spleen lymphocyte suspension was determined by flow cytometry CD4 and CD8 T cells percentage. Indirect ELISA method-specific IFN-γ and IL-10 levels, and serum specific antibody titers. CCK-8 detection spleen lymphocyte proliferation results using the stimulation index (Stimulation index, SI) SI = the experimental wells A / A control well. Stimulation index lt; Annexin V-PI kit to detect whether apoptosis. Results: (1) normal group with the the VD group of weight without significant difference (P gt; 0.05); VD group, VD-group of eight weeks, the average consumption of feed 12 Sunday, statistical analysis 8 weeks, 12 weeks No difference. Normal group VD group, VD-plasma albumin were 33.2 ± 1.04,32.4 ± 0.53,32.8 ± 0.67 (g / L), no statistically significant difference. VD level of the normal group and VD group plasma no statistically significant differences, but the VD-plasma VD decreased to less than 20% of the normal, suggesting that the lack of mouse VD model was successfully established; After six weeks, the group I and group II VD levels 18.46 ± 1.53,58.46 ± 5.53 (nmol / L) (P lt; 0.001). Measured sub murine plasma calcium levels were 2.32 ± 0.087,2.49 ± 0.144,1.73 ± 0.091 (mmol / L), plasma phosphorus levels were 2.20 ± 0.071,2.41 ± 0.042,1.77 ± 0.091 (mmol / L) after The statistical analysis of the plasma calcium and phosphorus in the three groups were statistically significant (P lt; 0.05), the normal group and the VD group was no significant difference (P gt; 0.05), VD-group and the other two groups have significant differences ( P lt; 0.05). 5.BCG stimulate normal group VD group and VD-group after six weeks, the spleen lymphocytes in CD4 cell subsets percentage were 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 rise especially bigger CD8 rise. 6 plasma IFN-γ were 375.40 ± 13.11,301.96 ± 23.60,478.43 ± 17.50 (pg / ml), plasma IL-10 were 27.35 ± 2.81,28.55 ± 0.72,21.47 ± 1.34 (pg / ml), VD- group significantly increased compared to the other two groups of plasma IFN-γ and IL-10 significantly reduced. Sera of 7.VD-group-specific anti-TB-PPD antibodies was significantly higher than the normal group and VD group. 8. Spleen lymphocytes value-added stimulation index were 1.47 ± 0.12,1.15 ± 0.11,0.65 ± 0.16, VD-suppressed splenic T lymphocyte proliferative capacity in TB-PPD stimulation, VD-group of lymphocytes appear significantly apoptosis. Conclusion: by controlling the food and light, successfully established VD lack of an animal model of BALB / c mice. VD natural sources, the role of ultraviolet light than diet role important, but directly supplement the the VD effect is significant. 3.VD deficiency can cause a range of diseases, rickets symptoms in this experiment. 4.VD lack of significant changes in the immune system response characteristics of BCG stimulation with VD, suggesting that the lack of easily lead to TB-related. 5.VD to play an important etiology role in innate immunity, while increasing immune tolerance, reduce the autoimmune damage. This provides a new way of thinking for the clinical treatment of tuberculosis infections and autoimmune diseases.
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