免疫調節(jié)劑對家兔皮膚結核模型液化進程及結核菌載量的影響
發(fā)布時間：2018-08-20 12:34
【摘要】：目的：評價免疫調節(jié)劑重組人干擾素-γ(rIFN-γ)、重組人白細胞介素-2(rIL-2)、地塞米松及環(huán)磷酰胺(CTX)分別對家兔皮膚結核結節(jié)形成發(fā)展過程(特別是結節(jié)液化發(fā)生、發(fā)展過程)及液化物質中結核菌載量的影響,初步建立家兔皮膚結核液化模型藥物評價方法,從而為結核病灶液化及空洞形成發(fā)展機制的研究及相關治療藥物的評價提供研究思路及方法。方法：隨機將家兔分為六組,即rIFN-γ組、rIL-2組、高劑量地塞米松(DH)組、低劑量地塞米松(DL)組、CTX組及生理鹽水對照組。各組分別以一定劑量和用法對健康家兔給予干預。免疫調節(jié)劑干預第17天,用5×106CFU的牛分枝桿菌減毒株BCG對家兔腹背部進行皮內注射,建立家兔皮膚結核模型。在其后的持續(xù)干預過程中,動態(tài)觀察各組家兔皮膚結核結節(jié)形成發(fā)展過程,記錄結節(jié)肉芽腫、液化、破潰、愈合的時間和各時期結節(jié)的形態(tài)和大小,并觀察特定時期各組家兔皮膚結節(jié)的病理形態(tài)；計數(shù)各組家兔皮膚結核結節(jié)液化高峰期的液化物質中的結核菌載量;ELISA法檢測各組家兔血清中細胞因子IFN-γ、IL-2.IL-4.IL-17分泌水平；綜合分析細胞因子水平對結核結節(jié)形成發(fā)展的影響及細胞因子水平與結核結節(jié)液化物質中結核菌載量的關系。 結果： 1.各組家兔皮膚結核結節(jié)的發(fā)展變化有差異,具體表現(xiàn)為：各組家兔結核結節(jié)發(fā)展的各時期(肉芽腫、液化,破潰,愈合)時間不同,各組在各時期的結核結節(jié)體積大小不同： rIL-2組家兔皮肽結核結節(jié)的液化進程最快,在BCG接種后第8天即開始液化,破潰最早,在BCG接種后第11天結節(jié)發(fā)生破潰,并出現(xiàn)兩次液化高峰,BCG接種后第13天即達到第一次液化高峰。液化時結節(jié)體積在各組中最大,第一次液化高峰時達到3289±354mm3,明顯大于對照組結節(jié)體積2563±17mm3(P0.05)。結節(jié)消退速度快,愈合開始時間最早,在BCG接種后第17天便開始第一次液化后的愈合。rIFN-γ組的結核結節(jié)各時期發(fā)生時間和結節(jié)體積僅次于rIL-2組,也出現(xiàn)兩次液化高峰。 環(huán)磷酰胺組(CTX)結核結節(jié)各時期的發(fā)生時間與對照組無差異,結節(jié)體積小于對照組。低劑量地塞米松(DL)組的皮膚結核結節(jié)體積最小,肉芽腫時期最大體積為751±153mm3,顯著小于對照組結節(jié)體積1699±149mm3(P0.05)。液化高峰時結節(jié)體積(601±297mm3)也顯著小于對照組(2563±17mm3)(P)0.05),且液化過程不明顯。高劑量地塞米松(DH)組未形成明顯的肉芽腫結節(jié),且未觀察到液化現(xiàn)象。 2.各組家兔皮膚結核結節(jié)液化高峰期液化物質中的結核菌載量不同： rIL-2組結核菌載量為各組中最低,第一次液化高峰時結核菌載量為3.0×106CFU/g,顯著低于對照組結核菌載量(6.9x106CFU/g),第二次液化高峰時結核菌載量(1.7×105CFU/g)顯著低于第一次液化高峰時的載量。低劑量地塞米松(DL)組結核菌載量(2.8×107CFU/g)顯著高于對照組,為各組中最高。 3.各組家兔皮膚結核結節(jié)發(fā)展變化的不同時期內血清細胞因子IFN-γ、IL-2、IL-4、IL-17的分泌水平不同： γIFN-γ組在肉芽腫期的血清IFN-γ水平(1706.3±104.3pg/ml)和液化高峰期的水平(2662.9±208.5pg/ml)均顯著增高,高于給藥前水平(866.2±29pg/ml),且液化高峰期水平(2662.9±208.5pg/ml)高于對照組液化高峰期的水平(964.9±51.7pg/m1)。 rIL-2組和rIFN-γ組的血清IL-2水平在肉芽腫期(rIL-2組：289.1±14.5pg/ml；rIFN-γ組：211.1±17.6pg/m1)、液化高峰期(rIL-2組：318±25.7pg/ml;rIFN-γ組：297.9±23.2pg/m1)和愈合期(rIL-2組：205.7±9.9pg/ml;rIFN-γ組：231.6±19.8pg/m1)均顯著增高,高于各組給藥前水平(rIL-2組：103.5±3.9pg/ml;rIFN-γ組：79.2±2.0pg/ml),且液化高峰期的水平最高(rIL-2組：318±25.7pg/ml;rIFN-γ組：297.9±23.2pg/ml),均高于對照組液化高峰期的水平(201±5.9pg/m1)。 對照組的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,顯著高于給藥前水平(50.6±0.5pg/ml)。 各組的血清IL-17水平在液化高峰期時均增高,其中rIFN-γ組在液化高峰期時的IL-17水平(130.1±9.6pg/ml)顯著高于給藥前水平(54.4±2.3pg/ml)。 4.細胞因子水平與結核結節(jié)大小及細胞因子水平與結核結節(jié)液化物質中細菌載量的相關性分析顯示血清IL-2水平與結核結節(jié)大小呈正相關(r2=0.748,P=0.058),與液化高峰期液化物質中結核菌載量呈反相關(r2=0.875,P=0.020)。 結論： 1.不同免疫調節(jié)劑對結核結節(jié)形成發(fā)展過程(特別是結節(jié)液化發(fā)生、發(fā)展過程)產生不同影響,對病灶內結核菌的存活也產生不同影響。 2.白細胞介素-2顯著促進結核病灶液化的發(fā)生和發(fā)展,并促進結核病灶內結核菌載量的下降。地塞米松抑制結核病灶液化的發(fā)生和發(fā)展,并有利于結核菌在病灶中的存活。 3.初步建立了家兔皮膚結核液化模型藥物評價方法。
[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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
本文編號：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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
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