TMB-8对小鼠体外免疫应答和迟发型超敏反应的影响

发布时间：2018-05-05 19:33

本文选题：TMB-8 + 淋巴细胞　；参考：《暨南大学》2007年硕士论文

【摘要】： 目的：研究胞内钙离子释放阻断剂8-(N，N-二乙胺)辛基-3，4，5-三甲氧基苯甲酸酯([8-(Diethylamino)Octyl-3，4，5-Trimethoxybenzoate]，TMB-8)对小鼠体外免疫应答和体内迟发型超敏反应的影响，评价TMB-8的免疫抑制效应。方法： 1．分别以刀豆蛋白A(Concanavalin A，Con A)和／或2-丁酸佛波醇酯(Phorbol12，13-dibutyrate，PDB)加离子霉素(Ionomycin，Ion)作为刺激原，以流式细胞术为基础技术，分析TMB-8对小鼠体外淋巴细胞行为的影响：利用荧光标记抗体染色技术检测淋巴细胞早期活化标记物CD69的表达百分率；以活体染料二乙酰羧基荧光素-琥珀酰亚胺酯(Carboxyfluorescein diacetate succinimidyl ester，CFDA-SE)标记，分析TMB-8对淋巴细胞增殖的影响；以碘化丙啶(Propidium iodide，PI)染色分析细胞周期的分布情况。并以噻唑兰(Monotetrazolium，MTT)比色法测定TMB-8对脂多糖(Lipopolysaccharide，LPS)刺激B淋巴细胞增殖的影响。 2．通过收集ConA作用下的淋巴细胞培养上清，以流式微球技术(Cytometric bead array,CBA)检测分析TMB-8对该上清中Th1／Th2型细胞因子(IL-2，IFN-γ，TNF-α，IL-4，IL-5)分泌的影响。 3．利用Fluo-4／AM染色，结合流式细胞术检测TMB-8对Con A和／或Ion作用下小鼠淋巴细胞内Ca~(2+)浓度的影响。 4．建立动物模型，，采用二硝基氟苯(Dinitrofluorobenzene，DNFB)腹部致敏、耳廓激发的方法建立小鼠迟发型超敏反应(Delayed type hypersensitivity，DTH)模型。以耳厚差、耳重差及左耳组织切片的病理变化观察TMB-8对小鼠DTH的影响；同时以胸腺指数、脾脏指数，分析TMB-8对DTH小鼠淋巴器官的影响；分离DTH小鼠的脾脏、淋巴结、胸腺分别进行细胞增殖、周期和凋亡的检测，初步评价TMB-8在体内的免疫学效应。结果： 1．在所设的浓度下，TMB-8对静息淋巴细胞无明显影响。在Con A刺激下，四个浓度的TMB-8对CD3~+淋巴细胞的CD69~+表达均有抑制作用(P＜0.05)，且对CD3~-淋巴细胞的CD69~+表达也有明显的抑制作用(P＜0.01)。对Con A或(PDB+Ion)刺激48h和72h下的淋巴细胞增殖呈明显抑制效应(P＜0.01)。细胞周期分析结果表明TMB-8能抑制细胞进入S期。MTT比色法测定显示TMB-8对B淋巴细胞增殖呈抑制效应。 2．TMB-8对Con A活化的淋巴细胞培养上清中Th1型(IL-2，IFN-γ，TNF-α)和Th2型(IL-4，IL-5)细胞因子的分泌均有抑制作用(P＜0.05)。 3．TMB-8对ConA或Ion所诱导的细胞内Ca~(2+)浓度的升高有抑制作用，且随着时间的延长，这种抑制作用越来越明显。 4．TMB-8能够明显抑制DTH小鼠增加的左耳厚度差(P＜0.01)和重量差(P＜0.05)，并可减低DTH小鼠的脾脏指数和胸腺指数(P＜0.05)。同时组织切片显示TMB-8可明显抑制左耳组织水肿和炎细胞浸润。结合体外实验结果表明，TMB-8对DTH小鼠淋巴细胞增殖有明显的抑制作用，主要抑制细胞进入S期，并且能抑制DTH小鼠胸腺细胞的凋亡(P＜0.01)。结论： TMB-8在体外能抑制淋巴细胞的早期活化、增殖，并且能抑制淋巴细胞进入细胞分裂周期，说明其具有免疫调节作用。同时研究还证实它能抑制ConA介导的淋巴细胞Th1型和Th2型细胞因子的分泌。TMB-8能下调Con A或Ion诱导的细胞内Ca~(2+)浓度的上升，推测这是TMB-8发挥免疫抑制作用的基础。在这些研究结果的基础上，通过建立小鼠DTH动物模型，证实TMB-8在体内能抑制T淋巴细胞介导的皮肤炎症反应。
[Abstract]:Purpose :

The effects of 8 - ( N , N - Diethylamine ) octyl - 3 , 4 , 5 - Trimethoxybenzoate ( TB8 - 3 , 4 , 5 - Trimethoxybenzoate ) on the in vitro immune response and delayed hypersensitivity in mice were investigated .

Method :

1 . Ionomycin ( Ion ) was used as a stimulus to detect the expression of CD69 in lymphocytes in vitro by fluorescence labeled antibody staining .
The effects of T lymphocyte proliferation on lymphocyte proliferation were analyzed by labeling with biacetyl carboxyfluorescein - succinimide ester ( CFDA - SE ) in vivo .
The distribution of cell cycle was analyzed by propidium iodide ( PI ) staining , and the effects of IL - 8 on the proliferation of B lymphocytes stimulated by lipopolysaccharide ( LPS ) were determined by MTT assay .

2 . By collecting the lymphocyte culture supernatant under the action of concanavalin , the influence on secretion of Th1 / Th2 cytokines ( IL - 2 , IFN - 纬 , TNF - 伪 , IL - 4 , IL - 5 ) in the supernatant was detected by Cytometric bead array ( CBA ) .

3 . Fluo - 4 / AM staining combined with flow cytometry was used to detect the concentration of Ca ~ ( 2 + ) in lymphocytes of mouse lymphocytes induced by Con A and / or Ion .

4 . To establish an animal model , the delayed type hypersensitivity ( dth ) model was established by using dinitrobenzene ( DNFB ) abdominal sensitization and auricle excitation method .
At the same time , thymus index and spleen index were used to analyze the effect on lymphatic organs of mice .
The spleen , lymph nodes and thymus were isolated and the proliferation , cycle and apoptosis of the spleen , lymph nodes and thymus were detected , and the immunological effect in vivo was preliminarily evaluated .

Results :

1 . There was no significant effect on the expression of CD69 ~ + in CD3 ~ + lymphocytes under Con A stimulation ( P < 0 . 05 ) . There was a significant inhibitory effect on the expression of CD69 ~ + in CD3 ~ + lymphocytes ( P < 0.01 ) .

2 . The secretion of Th1 type ( IL - 2 , IFN - 纬 , TNF - 伪 ) and Th2 type ( IL - 4 , IL - 5 ) cytokines in the supernatant of Con A - activated lymphocytes was inhibited ( P < 0.05 ) .

3 . The increase of Ca ~ ( 2 + ) concentration in the cells induced by Con A or Ion has an inhibitory effect on the increase of intracellular Ca ~ ( 2 + ) concentration , and this inhibition is more and more obvious with the extension of time .

4 . The results showed that the proliferation of lymphocyte proliferation was inhibited significantly , and the apoptosis was inhibited significantly ( P & lt ; 0.01 ) .

Conclusion :

It can inhibit the proliferation of lymphocytes and inhibit the secretion of Th1 and Th2 cytokines in lymphocytes induced by Con A or Ion . The results of these studies show that it can downregulate the concentration of intracellular Ca ~ ( 2 + ) induced by Con A or Ion . It is speculated that this is the basis for the immunosuppressive effect .

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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