| Objective:This study was to gain an insight into the influence of vitamin D deficiency on thedistribution of spinal tuberculosis in peripheral blood CD4+and CD8+T lymphocytesubgroups and regulatory lymphocytes Treg. And to observe the influence of vitamin Ddeficiency on the expression of IL-2, IL-10in serum. And to observed the affect of vitaminD deficiency on the expression of IFN-γ and TGF-β, IL-10and TNF-α in spinal tuberculouslesions. We would like to discuss a possible mechanism of vitamin D deficiency on spinaltuberculosis immune function. The results were supposed to provide a basis for furtherresearch in vivo.Methods:1. The selection of subjectsWe selected142cases of spinal tuberculosis patients who received treatment inSouthwest Hospital Orthopedic from August2008to December2011as the study group.Inclusion criteria for initial tuberculous, normal nutritional status, long-term residence inChongqing, no history of foreign residence, Han population, age of16-65years old,vaccination history, etc. And then we selected50cases of lumbar disc patients whose agedistribution, the nutritional status were similar to the study group as the control group.Next we measured their serum albumin, serum calcium, phosphorus level to rule out theinfluencing factors of nutritional status or calcium and phosphate metabolismabnormalities to VD.2. Determination of VD level and grouping of study groupWe tested the level of serum VD by ELISA Kit in both the study group and controlgroup. The study group were divided into two groups, one is VD normal group which VDlevel higher than50nmol/L, another is VD deficient group which VD is lower than50nmol/L. According to observation of the lesions in operation and the radiological characteristics the study groups were divided into hyperplastic type and caseous type. Thedifference of lesion types constitute ratio between the two types was analysed by Chi-squared test.3. Determination and analysis of peripheral blood immune parametersThe distribution of CD4+and CD8+T lymphocyte subgroups and regulatorylymphocytes CD4+CD25+Foxp3+Treg were measured by flow cytometry. The expressionof the cytokines IL-2, IL-10was tested by ELISA Kit. One-Way ANOVA was designed toanalyse difference of each parameters between the groups.4. Determination and analysis of local lesions immune parametersLesion specimens were obtained by surgery from the study group. The pus wassmeared and stained with HE, and peformed immunohistochemistry for IL-10and TNF-α.The sequestrum was paraffin-embedded and made into pathological sections and stainedwith HE for observation. The abscess wall granuloma was paraffin-embedded and madeinto pathological sections and stained with HE and peformed immunohistochemistry forIFN-γ, TGF-β, IL-10, TNF-α. We observed the morphological characteristics of the locallesion and analysed the differential expression of various cytokines in the two groups oflesions.Results:1. In the142cases of the study group, there are59cases whose serum VD level wereabove50nmol/L as VD normal group and83cases whose serum VD level were below50nmol/L as VD deficient group. All the experimental individual were measured serumalbumin, serum calcium, phosphorus level in the normal range. It would exclude theinfluencing factors of nutritional status or calcium and phosphate metabolismabnormalities to VD.2. The level of serum VD was59.7±5.72nmol/L in control group（n=50）and56.9±5.48nmol/L in VD normal group（n=59）and41.5±4.67nmol/L in VD deficientgroup. There is no significant difference in serum VD level between the VD normal groupand control group. However, Serum VD level of VD deficient group was significantlylower than the other two groups （P <0.05）. The proportion of caseous type wassignificantly higher in VD deficient group85.5%（71/83）than in VD normal group37.3%（22/59）（P＜0.01）. 3. The result of three-channel flow cytometry:①The percentage of CD4+T lymphocyte subgroups is significantly higher in VDdeficient group（38.18±3.64）%than in VD normal group（35.92±3.47）%or in controlgroup（35.61±2.95）%（P＜0.05）. The percentage of CD8+T lymphocyte subgroups is alsosignificantly higher in VD deficient group（43.16±2.76）%than in VD normal group（31.21±1.60）%or in control group（28.93±1.70）%（P＜0.01）. The CD4+/CD8+ratiois significantly lower in VD deficient group（0.90±0.08） than in VD normal group（1.26±0.10）or in control group（1.30±0.12）（P＜0.01）. There is no significant differencebetween VD normal group and control group.②The percentage of CD4+CD25+Foxp3+regulatory T cell subgroup within CD4+lymphocyte is significantly lower in VD deficient group（3.92±0.67）%than in VD normalgroup（6.03±0.69）%or in control group（5.41±0.86）%（P＜0.05）.The result of ELISA Kit:①The level of serum IL-2is significantly higher in VD deficient group67.42±6.23ng/L than in VD normal group58.63±5.87ng/L or in control group56.35±7.55ng/L（P＜0.05）.②The level of serum IL-10is significantly lower in VD deficient group12.63±2.20ng/L than in VD normal group20.05±3.15ng/L or in control group18.48±2.90ng/L（P＜0.01）.4.The result of immunohistochemistry of lesion①The expression ofIL-10in pus smear is significantly lower in VD deficient groupthan in VD normal group（P＜0.05）. Meanwhile, the expression of TNF-α in pus smear issignificantly higher in VD deficient group than in VD normal group（P＜0.05）.②The expression of IL-10, TGF-β in the abscess wall granuloma paraffin sections issignificantly lower in VD deficient group than in VD normal group（P＜0.05）.However,the expression of IFN-γ, TNF-α in the abscess wall granuloma paraffin sections issignificantly higher in VD deficient group than in VD normal group（P＜0.05）Conclusion:1. The significantly higher proportion of caseous type in VD deficient group suggestthat VD deficiency would be the reason why spinal tuberculosis turning to the poorprognosis. 2. VD deficiency cause of apparent disorder of CD4+and CD8+T lymphocytesubgroups distribution, and imbalance of the Th1/Th2ratio which were representative ofIL-2/IL-10, significantly low percentage of CD4+CD25+Foxp3+Treg within CD4+lymphocyte. It breaked the balance of protective immunity and immune injury. It probablybe the origin of spinal tuberculosis susceptibility and severity of exacerbations.3. In local lesion, VD deficiency lead to lower expression of IL-10, TGF-β and higherexpression of IFN-γ, TNF-α. It may cause tuberculosis local allergic reaction to the moreserious results. Meanwhile, VD sufficiency may prevent lesion from excessive self-injury,and lead the disease to the good side, and make the cellular immunity to show a moderateprotective and immune tolerance in local lesions... |
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