免疫调节剂对家兔皮肤结核模型液化进程及结核菌载量的影响
发布时间：2018-08-20 12:34
【摘要】：目的：评价免疫调节剂重组人干扰素-γ(rIFN-γ)、重组人白细胞介素-2(rIL-2)、地塞米松及环磷酰胺(CTX)分别对家兔皮肤结核结节形成发展过程(特别是结节液化发生、发展过程)及液化物质中结核菌载量的影响,初步建立家兔皮肤结核液化模型药物评价方法,从而为结核病灶液化及空洞形成发展机制的研究及相关治疗药物的评价提供研究思路及方法。方法：随机将家兔分为六组,即rIFN-γ组、rIL-2组、高剂量地塞米松(DH)组、低剂量地塞米松(DL)组、CTX组及生理盐水对照组。各组分别以一定剂量和用法对健康家兔给予干预。免疫调节剂干预第17天,用5×106CFU的牛分枝杆菌减毒株BCG对家兔腹背部进行皮内注射,建立家兔皮肤结核模型。在其后的持续干预过程中,动态观察各组家兔皮肤结核结节形成发展过程,记录结节肉芽肿、液化、破溃、愈合的时间和各时期结节的形态和大小,并观察特定时期各组家兔皮肤结节的病理形态；计数各组家兔皮肤结核结节液化高峰期的液化物质中的结核菌载量;ELISA法检测各组家兔血清中细胞因子IFN-γ、IL-2.IL-4.IL-17分泌水平；综合分析细胞因子水平对结核结节形成发展的影响及细胞因子水平与结核结节液化物质中结核菌载量的关系。 结果： 1.各组家兔皮肤结核结节的发展变化有差异,具体表现为：各组家兔结核结节发展的各时期(肉芽肿、液化,破溃,愈合)时间不同,各组在各时期的结核结节体积大小不同： rIL-2组家兔皮肽结核结节的液化进程最快,在BCG接种后第8天即开始液化,破溃最早,在BCG接种后第11天结节发生破溃,并出现两次液化高峰,BCG接种后第13天即达到第一次液化高峰。液化时结节体积在各组中最大,第一次液化高峰时达到3289±354mm3,明显大于对照组结节体积2563±17mm3(P0.05)。结节消退速度快,愈合开始时间最早,在BCG接种后第17天便开始第一次液化后的愈合。rIFN-γ组的结核结节各时期发生时间和结节体积仅次于rIL-2组,也出现两次液化高峰。 环磷酰胺组(CTX)结核结节各时期的发生时间与对照组无差异,结节体积小于对照组。低剂量地塞米松(DL)组的皮肤结核结节体积最小,肉芽肿时期最大体积为751±153mm3,显著小于对照组结节体积1699±149mm3(P0.05)。液化高峰时结节体积(601±297mm3)也显著小于对照组(2563±17mm3)(P)0.05),且液化过程不明显。高剂量地塞米松(DH)组未形成明显的肉芽肿结节,且未观察到液化现象。 2.各组家兔皮肤结核结节液化高峰期液化物质中的结核菌载量不同： rIL-2组结核菌载量为各组中最低,第一次液化高峰时结核菌载量为3.0×106CFU/g,显著低于对照组结核菌载量(6.9x106CFU/g),第二次液化高峰时结核菌载量(1.7×105CFU/g)显著低于第一次液化高峰时的载量。低剂量地塞米松(DL)组结核菌载量(2.8×107CFU/g)显著高于对照组,为各组中最高。 3.各组家兔皮肤结核结节发展变化的不同时期内血清细胞因子IFN-γ、IL-2、IL-4、IL-17的分泌水平不同： γIFN-γ组在肉芽肿期的血清IFN-γ水平(1706.3±104.3pg/ml)和液化高峰期的水平(2662.9±208.5pg/ml)均显著增高,高于给药前水平(866.2±29pg/ml),且液化高峰期水平(2662.9±208.5pg/ml)高于对照组液化高峰期的水平(964.9±51.7pg/m1)。 rIL-2组和rIFN-γ组的血清IL-2水平在肉芽肿期(rIL-2组：289.1±14.5pg/ml；rIFN-γ组：211.1±17.6pg/m1)、液化高峰期(rIL-2组：318±25.7pg/ml;rIFN-γ组：297.9±23.2pg/m1)和愈合期(rIL-2组：205.7±9.9pg/ml;rIFN-γ组：231.6±19.8pg/m1)均显著增高,高于各组给药前水平(rIL-2组：103.5±3.9pg/ml;rIFN-γ组：79.2±2.0pg/ml),且液化高峰期的水平最高(rIL-2组：318±25.7pg/ml;rIFN-γ组：297.9±23.2pg/ml),均高于对照组液化高峰期的水平(201±5.9pg/m1)。 对照组的IL-4水平在液化高峰期(91.2±10.1pg/m1)和愈合期(87.7±3.1pg/ml)增高,显著高于给药前水平(50.6±0.5pg/ml)。 各组的血清IL-17水平在液化高峰期时均增高,其中rIFN-γ组在液化高峰期时的IL-17水平(130.1±9.6pg/ml)显著高于给药前水平(54.4±2.3pg/ml)。 4.细胞因子水平与结核结节大小及细胞因子水平与结核结节液化物质中细菌载量的相关性分析显示血清IL-2水平与结核结节大小呈正相关(r2=0.748,P=0.058),与液化高峰期液化物质中结核菌载量呈反相关(r2=0.875,P=0.020)。 结论： 1.不同免疫调节剂对结核结节形成发展过程(特别是结节液化发生、发展过程)产生不同影响,对病灶内结核菌的存活也产生不同影响。 2.白细胞介素-2显著促进结核病灶液化的发生和发展,并促进结核病灶内结核菌载量的下降。地塞米松抑制结核病灶液化的发生和发展,并有利于结核菌在病灶中的存活。 3.初步建立了家兔皮肤结核液化模型药物评价方法。
[Abstract]:AIM: To evaluate the effects of recombinant human interferon-gamma (rIFN-gamma), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) on the formation and development of tuberculous nodules (especially the occurrence and development of tuberculous nodules) in rabbit skin and the load of tuberculous bacteria in the liquefied substances, respectively. Methods: Rabbits were randomly divided into six groups: rIFN-gamma group, rIL-2 group, high-dose dexamethasone (DH) group, low-dose dexamethasone (DL) group, CTX group and normal saline control group. On the seventeenth day after the intervention of immunomodulators, the rabbit skin tuberculosis model was established by intradermal injection of 5 *106 CFU attenuated Mycobacterium bovis strain BCG into the abdomen and back of rabbits. The formation and development of skin tuberculosis nodules were observed dynamically during the continuous intervention. The morphology and size of granuloma, liquefaction, ulceration, healing time and nodules in each period were recorded, and the pathological morphology of skin nodules in each group was observed; the tuberculosis bacterial load in the liquefied substances of skin tuberculosis nodules in each group was counted; the serum cytokines IFN-gamma, IL-2.IL-4 in each group were detected by ELISA. IL-17 secretion level; comprehensive analysis of cytokine level on tuberculosis nodule formation and development and cytokine level and tuberculosis nodule liquefaction substance in the relationship between tuberculosis bacterial load.
Result:
1. The development and change of tuberculous nodules in skin of rabbits in each group are different. The specific manifestations are as follows: the development time of tuberculous nodules in each group is different (granuloma, liquefaction, ulceration, healing), and the volume and size of tuberculous nodules in each group are different in each period:
In the rIL-2 group, the nodules began to liquefy at the 8th day after BCG inoculation. The nodules began to liquefy at the earliest time. On the 11th day after BCG inoculation, the nodules broke and had two peaks of liquefaction. On the 13th day after BCG inoculation, the nodules reached the first liquefaction peak. The nodule volume in rIFN-gamma group was significantly larger than that in rIL-2 group (P 0.05).
The tuberculous nodule volume of low dose dexamethasone (DL) group was the smallest, and that of granuloma group was 751 (+ 153mm3), which was significantly smaller than that of control group (1699 (+ 149mm3) (P 0.05). The nodule volume of low dose dexamethasone (DL) group was (601 < 297) at liquefaction peak. Mm3 was also significantly lower than that of control group (2563
2. in the peak period of liquefaction of skin tuberculous nodules in different groups of rabbits, the amount of Mycobacterium tuberculosis in liquefied substances varied.
The load of tuberculosis bacteria in rIL-2 group was the lowest in each group. At the first liquefaction peak, the load of tuberculosis bacteria was 3.0 *106 CFU/g, which was significantly lower than that in control group (6.9x106 CFU/g). At the second liquefaction peak, the load of tuberculosis bacteria (1.7 *105CFU/g) was significantly lower than that at the first liquefaction peak. 07CFU/g) was significantly higher than that in the control group, the highest among all groups.
3. The levels of serum cytokines IFN-gamma, IL-2, IL-4 and IL-17 were different in different stages of tuberculous nodule development in each group.
The levels of serum IFN-gamma in granulomatous stage (1706.3+104.3 pg/ml) and peak liquefaction stage (2662.9+208.5 pg/ml) in gamma-IFN-gamma group were significantly higher than those before administration (866.2+29 pg/ml), and the peak liquefaction stage (2662.9+208.5 pg/ml) was higher than that in control group (964.9+51.7 pg/ml).
Serum levels of IL-2 in rIL-2 group and rIFN-2 group and rIFN-gamma group were at granuloma stage (rIL-2 group: 289.1 + 14.5 pg / ml; rIFN-gamma group: 211.1 + 17.6 pg / ml 1), liquefaction peak (rIL-2 group: 318 + 25.7 pg / ml; rIFN-gamgroup: 297.9 + 23.2 pg / m1) and healstage (rIL-2 group: 205.7 + 9.7 + 9.9.9 9 9 pg / ml; rIFN-gamgroup: 211.1 1 1 1 + 17.1 + 17.6 pg / ml), liquliquliquefpeak (rIL-2 group: 318 + 25.8 + 25.7 7 7.7 It was significantly higher than that of the water before administration in each group. The levels of serum levels in rIL-2 group (103.5+3.9 pg/ml) and rIFN-gamma group (79.2+2.0 pg/ml) were the highest (318+25.7 pg/ml in rIL-2 group and 297.9+23.2 pg/ml in rIFN-gamma group), which were higher than those in control group (201+5.9 pg/m1).
The level of IL-4 in the control group was significantly higher at the peak liquefaction stage (91.2+10.1 pg/m1) and the healing stage (87.7+3.1 pg/ml) than that before administration (50.6+0.5 pg/ml).
The serum levels of IL-17 in each group increased during the peak period of liquefaction, and the levels of IL-17 in rIFN-gamma group at the peak period of liquefaction (130.1+9.6 pg/ml) were significantly higher than those before administration (54.4+2.3 pg/ml).
4. Correlation analysis of cytokine level with tuberculosis nodule size and cytokine level with tuberculosis nodule liquefaction substance bacterial load showed that serum IL-2 level was positively correlated with tuberculosis nodule size (r2 = 0.748, P = 0.058), and was inversely correlated with tuberculosis bacterial load in liquefaction substance during liquefaction peak (r2 = 0.875, P = 0.020).
Conclusion:
1. Different immunomodulators have different effects on the formation and development of tuberculosis nodules, especially on the liquefaction and development of tuberculosis nodules, and on the survival of tuberculosis bacteria in the nodules.
2. Interleukin-2 significantly promotes the occurrence and development of tuberculosis lesion liquefaction, and promotes the decrease of tuberculosis bacteria load in the lesion. Dexamethasone inhibits the occurrence and development of tuberculosis lesion liquefaction, and is conducive to the survival of tuberculosis bacteria in the lesion.
3. a preliminary evaluation method for rabbit skin tuberculosis liquefaction model was established.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2193625
[Abstract]:AIM: To evaluate the effects of recombinant human interferon-gamma (rIFN-gamma), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) on the formation and development of tuberculous nodules (especially the occurrence and development of tuberculous nodules) in rabbit skin and the load of tuberculous bacteria in the liquefied substances, respectively. Methods: Rabbits were randomly divided into six groups: rIFN-gamma group, rIL-2 group, high-dose dexamethasone (DH) group, low-dose dexamethasone (DL) group, CTX group and normal saline control group. On the seventeenth day after the intervention of immunomodulators, the rabbit skin tuberculosis model was established by intradermal injection of 5 *106 CFU attenuated Mycobacterium bovis strain BCG into the abdomen and back of rabbits. The formation and development of skin tuberculosis nodules were observed dynamically during the continuous intervention. The morphology and size of granuloma, liquefaction, ulceration, healing time and nodules in each period were recorded, and the pathological morphology of skin nodules in each group was observed; the tuberculosis bacterial load in the liquefied substances of skin tuberculosis nodules in each group was counted; the serum cytokines IFN-gamma, IL-2.IL-4 in each group were detected by ELISA. IL-17 secretion level; comprehensive analysis of cytokine level on tuberculosis nodule formation and development and cytokine level and tuberculosis nodule liquefaction substance in the relationship between tuberculosis bacterial load.
Result:
1. The development and change of tuberculous nodules in skin of rabbits in each group are different. The specific manifestations are as follows: the development time of tuberculous nodules in each group is different (granuloma, liquefaction, ulceration, healing), and the volume and size of tuberculous nodules in each group are different in each period:
In the rIL-2 group, the nodules began to liquefy at the 8th day after BCG inoculation. The nodules began to liquefy at the earliest time. On the 11th day after BCG inoculation, the nodules broke and had two peaks of liquefaction. On the 13th day after BCG inoculation, the nodules reached the first liquefaction peak. The nodule volume in rIFN-gamma group was significantly larger than that in rIL-2 group (P 0.05).
The tuberculous nodule volume of low dose dexamethasone (DL) group was the smallest, and that of granuloma group was 751 (+ 153mm3), which was significantly smaller than that of control group (1699 (+ 149mm3) (P 0.05). The nodule volume of low dose dexamethasone (DL) group was (601 < 297) at liquefaction peak. Mm3 was also significantly lower than that of control group (2563
2. in the peak period of liquefaction of skin tuberculous nodules in different groups of rabbits, the amount of Mycobacterium tuberculosis in liquefied substances varied.
The load of tuberculosis bacteria in rIL-2 group was the lowest in each group. At the first liquefaction peak, the load of tuberculosis bacteria was 3.0 *106 CFU/g, which was significantly lower than that in control group (6.9x106 CFU/g). At the second liquefaction peak, the load of tuberculosis bacteria (1.7 *105CFU/g) was significantly lower than that at the first liquefaction peak. 07CFU/g) was significantly higher than that in the control group, the highest among all groups.
3. The levels of serum cytokines IFN-gamma, IL-2, IL-4 and IL-17 were different in different stages of tuberculous nodule development in each group.
The levels of serum IFN-gamma in granulomatous stage (1706.3+104.3 pg/ml) and peak liquefaction stage (2662.9+208.5 pg/ml) in gamma-IFN-gamma group were significantly higher than those before administration (866.2+29 pg/ml), and the peak liquefaction stage (2662.9+208.5 pg/ml) was higher than that in control group (964.9+51.7 pg/ml).
Serum levels of IL-2 in rIL-2 group and rIFN-2 group and rIFN-gamma group were at granuloma stage (rIL-2 group: 289.1 + 14.5 pg / ml; rIFN-gamma group: 211.1 + 17.6 pg / ml 1), liquefaction peak (rIL-2 group: 318 + 25.7 pg / ml; rIFN-gamgroup: 297.9 + 23.2 pg / m1) and healstage (rIL-2 group: 205.7 + 9.7 + 9.9.9 9 9 pg / ml; rIFN-gamgroup: 211.1 1 1 1 + 17.1 + 17.6 pg / ml), liquliquliquefpeak (rIL-2 group: 318 + 25.8 + 25.7 7 7.7 It was significantly higher than that of the water before administration in each group. The levels of serum levels in rIL-2 group (103.5+3.9 pg/ml) and rIFN-gamma group (79.2+2.0 pg/ml) were the highest (318+25.7 pg/ml in rIL-2 group and 297.9+23.2 pg/ml in rIFN-gamma group), which were higher than those in control group (201+5.9 pg/m1).
The level of IL-4 in the control group was significantly higher at the peak liquefaction stage (91.2+10.1 pg/m1) and the healing stage (87.7+3.1 pg/ml) than that before administration (50.6+0.5 pg/ml).
The serum levels of IL-17 in each group increased during the peak period of liquefaction, and the levels of IL-17 in rIFN-gamma group at the peak period of liquefaction (130.1+9.6 pg/ml) were significantly higher than those before administration (54.4+2.3 pg/ml).
4. Correlation analysis of cytokine level with tuberculosis nodule size and cytokine level with tuberculosis nodule liquefaction substance bacterial load showed that serum IL-2 level was positively correlated with tuberculosis nodule size (r2 = 0.748, P = 0.058), and was inversely correlated with tuberculosis bacterial load in liquefaction substance during liquefaction peak (r2 = 0.875, P = 0.020).
Conclusion:
1. Different immunomodulators have different effects on the formation and development of tuberculosis nodules, especially on the liquefaction and development of tuberculosis nodules, and on the survival of tuberculosis bacteria in the nodules.
2. Interleukin-2 significantly promotes the occurrence and development of tuberculosis lesion liquefaction, and promotes the decrease of tuberculosis bacteria load in the lesion. Dexamethasone inhibits the occurrence and development of tuberculosis lesion liquefaction, and is conducive to the survival of tuberculosis bacteria in the lesion.
3. a preliminary evaluation method for rabbit skin tuberculosis liquefaction model was established.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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