【作者】 冷红；

【导师】 郑志忠； 骆肖群； 马莉；

【作者基本信息】 复旦大学 ， 皮肤性病学， 2009， 博士

【摘要】 第一部分ERK1/2 MAPK和p38 MAPK通路在iC3b结合的单核细胞向树突状细胞分化过程中的作用目的：研究补体iC3b对人血前体单核细胞(Mo)分化的影响,进一步验证UVB诱导的补体iC3b沉积和CDla+树突状细胞(DC)消失之间的关系；探讨ERK1/2 (ERK44/42) MAPK和p38 MAPK信号传导途径是否参与此影响,相应的MAPK抑制剂是否能逆转这种影响；经iC3b及ERK1/2特异性抑制剂或p38特异性抑制剂预处理后的Mo对CD4+T细胞的作用是否与正常情况下分化的Mo不同。方法：1)从健康人外周血通过免疫磁珠法分离前体Mo。EAiC3b (IgM-plus iC3b-coated sheep erythrocyte, IgM以及iC3b包被的羊红细胞)与人血前体Mo以及IL-4、GM-CSF共同培养2天,并与EA(IgM coated sheep erythrocyte, IgM包被的羊红细胞)作对照,用流式细胞仪检测EAiC3b对前体Mo分化的影响(CD14+,CDla+); Western blot检测磷酸化ERK1/2、p38 MAPK的蛋白水平,ELISA法检测IL-10、IL-12p70水平。2)上述培养过程中加入ERK1/2 MAPK抑制剂(PD98059)、p38 MAPK抑制剂(SB203580)预处理后,磷酸化ERK1/2、p38 MAPK和IL-10、IL-12p70水平的水平,以及DC数量的变化。3)CD4+T细胞增殖实验：Mo经PD98059(或SB203580)和EAiC3b预处理后,与IL-4、GM-CSF共培养6天经60Co照射后作为刺激细胞,同时分离同种异体人外周血CD4+T作为反应细胞,共培养5天后,经3H—掺入法检测CD4+T细胞增殖反应。结果：1)相比EA组结果,EAiC3b的加入使Mo向CDla+DC方向的分化受到抑制,表现为CDla+的表达明显降低,磷酸化—ERK1／2 MAPK的表达增加,磷酸化—p38 MAPK的表达降低,IL-12p70的水平降低,IL-10的水平增加,CD4+T细胞增殖减弱。2)前过程中加入PD98059预处理后,相比EAiC3b结果,则促进了Mo向CDla+DC方向的分化成熟,表现为CDla+的表达明显增加,磷酸化—ERK1／2 MAPK的表达降低,IL-12p70的水平增高,IL-10的水平降低,CD4+T细胞增殖明显提高。3)细胞经SB203580预处理后,相比EAiC3b结果,Mo向CDla+DC方向的分化成熟被进一步抑制,表现为CDla+的表达降低,磷酸化—p38 MAPK的表达降低,IL-12p70的水平稍有降低,IL-10的水平略有增高,CD4+T细胞增殖有所下调。结论：1)紫外线引起的补体iC3b的沉积是引起CDla+DC消失的重要原因；2) ERK1／2 MAPK抑制剂PD98059可显著逆转这种现象,促进前体Mo向CDla+DC的分化,而p38 MAPK抑制剂SB203580则进一步抑制CDla+DC的分化成熟；3)经iC3b作用的Mo对CD4+T细胞的增殖能力明显减弱,PD预处理后可显著恢复,SB预处理后加强这种抑制。第二部分：ERK1/2 MAPK抑制剂PD98059可明显逆转UVB诱导的小鼠皮肤局部免疫抑制,未观察到SB203580对迟发性高敏反应的抗炎作用目的：在UVB诱导局部免疫抑制动物模型的基础上,观察外涂PD9059能否逆转该现象,以证实ERK1/2 MAPK在免疫抑制中的关键作用,为开发防光剂提供新的思路；在接触性高敏反应动物模型的基础上,观察外涂SB203580观察能否抑制炎症反应,为抗炎剂作基础。方法：建立UVB诱导局部免疫抑制的小鼠模型,即UVB 8 kJ/m2昭射Balb/c小鼠腹部去毛皮肤后3天,在相同区域外涂25μ10.5%DNFB致敏,5天后测量基础右耳廓厚度并在相同耳背处外涂10μ10.2%DNFB激发,24小时后测量耳廓厚度并取右耳组织做HE病理。PD研究：UVB曝光前1小时(A组),曝光同时(A组),曝光后6小时(C组)外涂PD98059,余步骤同前,检测耳肿反应及HE染色。SB研究：小鼠腹部去毛皮肤外涂不同浓度的SB203580,6小时后相同皮肤处外涂25μ10.5%DNFB致敏,5天后测量基础右耳廓厚度并在耳背皮肤外涂10μ10.2%DNFB激发,24小时后再次测量耳廓厚度并取耳组织做HE病理染色。各组均设对照。结果：PD研究：UVB 8 kJ/m2及其以上剂量可引起局部免疫抑制,在此基础上,光照后6小时外涂PD98059组(C组)可部分逆转UVB诱导的局部免疫抑制,表现为和对照组相比,耳肿反应有显著性差异(P<0.05),而照光前1小时(A组)和照光同时(B组)外涂PD98059组耳肿反应无统计学意义(P>0.05)；HE染色示真皮水肿增厚伴淋巴细胞的浸润。SB研究：和对照组相比,致敏前外涂不同浓度的SB203580对接触性高敏反应无明显改变(包括耳肿反应和病理HE染色)。结论：ERK1/2 MAPK通路在UVB诱导的皮肤免疫中起着关键作用,其抑制剂PD98059可显著逆转UVB诱导的免疫抑制,PD98059或类似物对UVB诱导的免疫抑制有预防保护作用；未观察到SB203580对迟发性性高敏反应的抗炎作用。更多还原

【Abstract】 Objective:As complement iC3b has been identified abundantly at the dermis-epidermis junction, and furthermore our previous study has demonstrated inhibition of monocyte-derived dendritic cell differentiation and interleukin-12 production by iC3b via a mitogen-activated protein kinase (MAPK) signaling pathway. The present study was thus supposed to investigate the roles of ERK1/2 and p38 MAPK cascades in the development of iC3b-coated-Mo into CD1a+DC, furthermore to test whether the disappearance of CD1a+DC can be reversed via their inhibitors, in addition, to explore the role of iC3b-coated-Mo with inhibitors pretreatment upon CD4+T proliferation compared with normal Mo.Methods:1) Mo was isolated with CD14+microbeads first, following incubation with EAiC3b (IgM-plus iC3b-coated sheep erythrocyte), IL-4 and GM-CSF for 2 days, then followed by detections of expression of CD14+and CD1a+via Flow cytometry, expression of phoso-ERK1/2 MAPK and phoso-p38 MAPK via Western blot, expression of IL-10 and IL-12 p70 via ELISA, compared with EA.2) When pretreatment with PD98059 (inhibitor for ERK1/2 MAPK) or SB203580 (inhibitor for p38 MAPK) Mo, prior to EaiC3b, also following 2-day incubation with IL-4 and GM-CSF, expression of CD 14+, CD1a+ via Flow cytometry, expression of phoso-ERK1/2 MAPK, phoso-p38 MAPK via Western blot, and expression of IL-10, IL-12 p70 via ELISA were all detected.3) PD98059 (or SB203580) pretreatment with Mo prior to EAiC3b, with GM-CSF and IL-4 incubated for 6 days, then cells were irradiated by 60Co as stimulating cells. CD4+T cells were isolated as response cells, and incubated with stimulating cells for 5 days. Then proliferation of CD4+T cells was detected via 3H-TdR. The controls were treated simultaneously.Results:Compared with EA results, maturation of CD1a+DC was inhibited by EAiC3b, that is, expressions of CD1a, phoso-p38 MAPK, IL-12p70 down-regulated, expressions of phoso-ERK1/2 MAPK, IL-10 up-regulated. When PD98059 pretreated, compared with the EAiC3b results, the inhibited maturation of imDC was reversed prominently, that is, expressions of CD1a, IL-12p70 up-regulated, and expressions of phoso-ERK1/2 MAPK, IL-10 down-regulated. When SB203580 pretreated, in contrast to the EAiC3b results, the inhibition of imDC maturation was enhanced, that is, expressions of CD1a, phoso-p38 MAPK, IL-12p70 down-regulated, and IL-10 level light up-regulated. Interestingly, the result of CD4+T proliferation (dpm) in iC3b-combined-Mo decreased dramatically compared with EA result. When PD pretreated, the result of CD4+T proliferation (dpm) was reversed obviously compared with EAC result. When SB pretreated, the result of CD4+T proliferation (dpm) showed enhanced inhibition compared with EAC result.Conclusion:1) Deposit of iC3b induced by UVB plays an important role in the disappearance of CDla+DC via ERK1/2 MAPK cascade; 2) pretreatment of the inhibitor PD98059 for ERK1/2 pathway could reversed the phenomenon dramatically, showing the promotion of imDC maturation, whereas the p38 MAPK specific inhibitor SB203580 pretreatd, the inhibition of imDC maturation was enhanced.3) Mo with iC3b pretreatment showed obvious downregulating potential upon CD4+T proliferation (dpm) compared with normal Mo. When PD pretreated, the potential could reversed to a significant extent.Objective:to explore the role of ERK1/2 MAPK in the mechanism of local UVB-induced immunosuppression, and to test the protective role of its specific inhibitor PD98059 against the phenomenon for the potential of suncreen design. In addition, to explore the anti-inflammatory effect of the p38 MAPK specific inhibitor SB203580 based on the mouse model of contact hypersensitivity.Methods:The mouse model of the local UVB-induced immuno—suppression was established first, that is,8 kJ/m2 of UVB was irradiated on the abdomen depilated skin on Balb/c mouse.3 days later the same area was sensitized by 25μl of 0.5% DNFB. Another 5 days later the baseline ear thickness of the right ear was measured and the dorsal skin of the same ear was challenged by 10μl of 0.2% DNFB. After 24 hours, the ear thickness was remeasured for ear swelling response and the same ear was biopsied for pathological HE stainings. PD study:based on the establised mouse model as described above, PD98059 was applied 1 hour before UVB exposure (group A), simultaneously (group B), and 6 hours after UVB exposure (group C) repectively. Evaluations were by ear swelling response and pathological HE staining. SB study:SB203580 was applied on the abdomen depilated skin on Balb/c mouse first. After 6 hours the same area was sensitized by 25μl of 0.5% DNFB.5 days later the baseline ear thickness of the right ear was measured and the dorsal skin of the same ear was challenged by 10μl of 0.2%DNFB. Another 24 hours later the ear thickness was remeasured for ear swelling response and the same ear was biopsied for pathological HE staining. All the controls were treated simultaneously.Results:PD study:based on the mouse model of local UVB-induced immunosuppression, PD98059 application 6 hours after UVB exposure (group C) showed reversal of immunosuppression, that is, ear swelling response is statistically significant compared with the control group P< 0.05), dermis thicken and edema with infiltration of lymphocytes. Group A and B showed no singificant. SB study:topical pretreatment with different doses of SB203580 based on the contact hypersensitivity mouse model failed to alleviate the degree of contact hypersensivity which were evaluated by ear swelling response and pathological HE staining, compared with the controls.Conclusion:ERK1/2 MAPK pathway plays a critical role in the nature of UVB-induced immunosuppression and its specific inhibitor PD98059 can reverse the immunosuppression prominently, and furthermore, PD98059 can protect against UVB-induced immunosuppression. The anti-inflammatory effect of the p38 MAPK specific inhibitor SB203580 based on contact hypersensivity was not observed in the present study.更多还原