补体C3、C3a受体在三氯乙烯致敏小鼠免疫性肾损伤中的作用研究
发布时间：2018-09-05 07:33
【摘要】：研究背景三氯乙烯(Trichloroethylene,TCE),作为一种常见金属洗涤剂、脂类萃取剂、涂料稀释剂广泛应用于电子、电镀、制药等行业。TCE可以引起职业接触人群发生职业性三氯乙烯药疹样皮炎(Occupational Dermatitis Medicamentosa-like of Trichloroethylene,ODMLT)。ODMLT可出现多系统、多脏器的损伤,其中部分患者可出现严重的肾脏损伤,严重威胁相关职业人群健康状况。对于ODMLT发病机制,目前研究发现除了迟发型IV型变态反应以外,补体系统在发病过程中可能也起到了重要的作用。在前期的动物实验研究中发现肾脏损伤中存在着补体系统的激活,补体C3a在致敏小鼠肾脏组织中有明显的沉积现象。C3a可与其特异性受体C3aR相结合,发挥扩张血管、趋化炎症细胞、调节炎症反应等功能。国内外研究发现C3a-C3aR系统在多种肾脏损伤中起到了十分重要作用,故深入研究补体系统在TCE致敏小鼠肾脏损伤中的作用对探讨ODMLT发病机制有着十分积极的意义。研究目的本研究通过检测小鼠肾脏中C3及C3aR的表达,使用C3aR特异性拮抗剂C3aRA(SB290157),来检测其对TCE致敏小鼠肾脏损伤的影响,揭示补体系统在TCE致敏小鼠免疫性肾损伤中的作用,进一步为探索职业性三氯乙烯药疹样皮炎(ODMLT)肾脏损伤机制提供依据。研究方法选取42只无特定病原体(SPF)级,雌性6-8周BALB/c小鼠,体重范围17.1-24.7(20.3±1.7)g。在适应性喂养一周后,根据随机分配原则将实验动物随机分为:空白对照组(5只)、溶剂对照组(5只)、TCE处理组(16只)和TCE+C3aRA联合处理组(16只)。参照BALB/c小鼠三氯乙烯致敏模型方法,在末次激发24h后,根据小鼠皮肤反应状况,按照皮肤致敏试验评分标准进行分组,当小鼠积分≥1分时,认为皮肤致敏阳性即分为致敏组,否则即判定为非致敏组,以此将处理组区分为致敏组和非致敏组。予末次激发后第3天,眼球采血及无菌取出肾脏组织,使用肾脏组织制备石蜡切片和冰冻切片。采用肌酐(CRE)、尿素氮(BUN)检测试剂盒,检测血清中肾功能相关指标CRE、BUN的水平;肾脏HE染色来观察肾脏病理变化状况;免疫组化检测肾脏组织中相关炎症因子的表达水平;实时荧光定量RT-PCR检测肾脏组织中C3、C3aR及炎症因子的逆转录水平;免疫荧光检测肾脏组织中足细胞蛋白Podocin的表达;Western Blotting检测肾脏组织中C3aR、Podocin蛋白的表达。研究结果(1)致敏情况:试验期间小鼠生长正常,各组间小鼠体重改变无明显差异。空白对照组和溶剂对照组小鼠皮肤未见明显变化,根据皮肤反应情况,TCE处理组致敏率7/16(43.75%),TCE+C3aRA处理组致敏率6/16(37.5%)。(2)肾脏病理结果显示:在空白对照组、溶剂对照组和所有非致敏组小鼠肾脏中未发现明显的病理改变。TCE致敏组肾脏中可见明显的炎性细胞浸润,肾小管结构改变,肾小管上皮细胞溶解现象。在C3aRA处理后致敏小鼠肾脏炎性细胞浸润明显减少、肾小管损伤减轻,肾脏损伤得到明显改善。(3)肾功能检测结果:溶剂对照组与各非致敏组之间CRE、BUN水平均未发现明显差异,TCE致敏组和TCE+C3aRA致敏组CRE、BUN均高于溶剂对照组和相应的非致敏组。但是,发现在C3aRA处理后,TCE+C3aRA致敏组CRE、BUN水平均明显低于TCE致敏组。(4)肾脏组织中C3aR测定结果:与空白对照组和溶剂对照组相比,TCE非致敏组C3aR在转录水平上均未见明显变化。与溶剂对照组和相应未致敏组比较,TCE致敏组小鼠肾脏组织中C3aR在转录水平上显著上升。Western Blotting结果也显示,空白对照组和溶剂对照组以及TCE非致敏组中C3aR蛋白的表达量较低,但是在TCE致敏组中C3aR蛋白的表达明显上升。(5)肾脏组织中C3测定结果:与空白对照组和溶剂对照组相比,TCE非致敏组在C3转录水平上未见明显变化,TCE致敏组肾脏组织中C3在转录水平上显著上升。(6)肾脏组织中细胞因子IL-1β、TNF-α测定结果:空白对照组和溶剂对照组与各非致敏组之间肾脏组织中IL-1β、TNF-α转录水平均未发现明显变化,TCE致敏组和TCE+C3aRA致敏组肾脏组织中IL-1β、TNF-α均高于溶剂对照组和相应非致敏组。但是发现在C3aRA处理后,TCE+C3aRA致敏组肾脏组织中IL-1β、TNF-α转录水平明显低于TCE致敏组。免疫组化也呈现相同的结果,在空白对照组和溶剂对照组中均未发现有IL-1β、TNF-α的明显表达,而在TCE致敏组和TCE+C3aRA致敏组肾脏组织中有明显的表达,且TCE+C3aRA致敏组IL-1β、TNF-α较的表达水平有所下降。(7)肾脏组织中足细胞Podocin蛋白测定结果:在空白对照组和溶剂对照组中Podocin蛋白可见正常表达,与空白对照组和溶剂对照组相比TCE致敏组和TCE+C3aRA致敏组小鼠肾脏Podocin蛋白的表达明显降低,但是与TCE致敏组相比,TCE+C3aRA致敏组Podocin蛋白表达水平有明显回升。Western Blotting结果也显示,与TCE致敏组相比,TCE+C3aRA致敏组与Podocin蛋白表达水平有明显回升。结论(1)TCE致敏小鼠肾脏中补体C3和C3aR表达明显上调,TCE致敏小鼠肾脏损伤中存在局部补体激活的现象。(2)补体C3aR信号通过上调炎症因子IL-1β和TNF-α的分泌,加重了TCE致敏小鼠免疫性肾脏损伤(3)TCE致敏小鼠免疫性肾损伤中Podocin蛋白的表达下调。(4)补体C3aR信号可能影响了TCE致敏小鼠肾脏中Podocin蛋白表达。
[Abstract]:Background Trichloroethylene (TCE), as a common metal detergent, lipid extractant, paint thinner is widely used in electronics, electroplating, pharmaceutical and other industries. TCE can cause occupational dermatitis dermatitis medicmentosa-like of Trichloroethylene (O. DMLT). ODMLT can cause multiple system and multiple organ injuries. Some of the patients may have severe kidney injuries, which seriously threaten the health of the occupational population. Current studies have found that complement system may play an important role in the pathogenesis of ODMLT in addition to delayed type IV allergy. C3a can be combined with its specific receptor C3aR to dilate blood vessels, chemotactic inflammatory cells, and regulate inflammation. It has been found that C3a-C3aR system can be used in a variety of kidney lesions at home and abroad. It is very important to study the role of complement system in the kidney injury of TCE sensitized mice. Objective To detect the expression of C3 and C3aR in the kidney of mice sensitized to TCE by detecting the expression of C3aR and C3aRA (SB290157), a specific antagonist of C3aR. The effect of complement system on renal injury in TCE-sensitized mice was revealed, which provided a basis for exploring the mechanism of renal injury in occupational trichloroethylene drug Eruption-like dermatitis (ODMLT). After one week of sexual feeding, the experimental animals were randomly divided into blank control group (5 rats), solvent control group (5 rats), TCE treatment group (16 rats) and TCE+C3aRA combined treatment group (16 rats). When the score of the mice was more than 1, the skin sensitized mice were divided into sensitized group and non-sensitized group. The treatment group was divided into sensitized group and non-sensitized group. On the third day after the last stimulation, the eyeballs were collected and the kidney tissues were aseptically removed. The paraffin sections and frozen sections were prepared using the kidney tissues. Creatinine (CRE), urea nitrogen (BUN) detection kit, detection of serum renal function related indicators CRE, BUN levels; kidney HE staining to observe renal pathological changes; immunohistochemical detection of renal tissue related inflammatory factors expression level; real-time quantitative RT-PCR detection of renal tissue C3, C3aR and inflammatory factors reverse transcription level; The expression of podocin in kidney tissue was detected by epidemic fluorescence, and the expression of C3aR and Podocin protein in kidney tissue was detected by Western Blotting. Results (1) Sensitization: The growth of mice was normal during the experiment, and there was no significant difference in body weight among the groups. The sensitization rate was 7/16 (43.75%) in the TCE group and 6/16 (37.5%) in the TCE + C3aRA group. (2) No obvious pathological changes were found in the kidneys of the blank control group, solvent control group and all non-sensitized mice. Renal tubular epithelial cell lysis. After C3aRA treatment, inflammatory cell infiltration in the kidney of sensitized mice was significantly reduced, renal tubular injury was alleviated, and renal injury was significantly improved. (3) Renal function test results: There was no significant difference in CRE and BUN levels between the solvent control group and the non-sensitized group, and there were no significant differences in CRE and BUN levels between the TCE sensitized group and the TCE+C3aRA sensitized group. However, the levels of CRE and BUN in TCE+C3aRA sensitized group were significantly lower than those in TCE sensitized group after C3aRA treatment. (4) The results of C3aR determination in renal tissue showed that there was no significant change in transcription level of C3aR in TCE sensitized group compared with blank control group and solvent control group. The expression of C3aR protein in kidney tissue of mice sensitized with TCE was significantly higher than that in non-sensitized group. Western Blotting results also showed that the expression of C3aR protein was lower in blank control group, solvent control group and TCE non-sensitized group, but the expression of C3aR protein was significantly increased in TCE sensitized group. Compared with the blank control group and solvent control group, there was no significant change in C3 transcription level in TCE non-sensitized group, and C3 transcription level in TCE sensitized group was significantly increased. (6) Determination of cytokines IL-1beta and TNF-alpha in renal tissue: IL-1beta and TNF-alpha in renal tissue between blank control group and solvent control group and non-sensitized group. The levels of IL-1 beta and TNF-alpha in renal tissue of TCE sensitized group and TCE+C3aRA sensitized group were higher than those of solvent control group and corresponding non-sensitized group. No significant expression of IL-1beta and TNF-a was found in the blank control group and solvent control group, but in the kidney tissues of TCE sensitized group and TCE+C3aRA sensitized group, and the expression levels of IL-1beta and TNF-a were decreased in TCE+C3aRA sensitized group. (7) Determination of podocin protein in the kidney tissues of blank control group and solvent The expression of Podocin protein in kidney of mice sensitized with TCE and sensitized with TCE+C3aRA was significantly lower than that of control group and solvent control group, but the expression of Podocin protein in TCE+C3aRA sensitized group was significantly higher than that of TCE sensitized group. Conclusion (1) The expression of complement C3 and C3aR in the kidney of TCE-sensitized mice was significantly up-regulated, and there was local complement activation in the kidney of TCE-sensitized mice. (2) Complement C3aR signaling aggravated the immunity of TCE-sensitized mice by up-regulating the secretion of inflammatory factors IL-1beta and TNF-alpha. (4) Complement C3aR signal may affect the expression of Podocin protein in the kidneys of TCE-sensitized mice.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.7
本文编号：2223606
[Abstract]:Background Trichloroethylene (TCE), as a common metal detergent, lipid extractant, paint thinner is widely used in electronics, electroplating, pharmaceutical and other industries. TCE can cause occupational dermatitis dermatitis medicmentosa-like of Trichloroethylene (O. DMLT). ODMLT can cause multiple system and multiple organ injuries. Some of the patients may have severe kidney injuries, which seriously threaten the health of the occupational population. Current studies have found that complement system may play an important role in the pathogenesis of ODMLT in addition to delayed type IV allergy. C3a can be combined with its specific receptor C3aR to dilate blood vessels, chemotactic inflammatory cells, and regulate inflammation. It has been found that C3a-C3aR system can be used in a variety of kidney lesions at home and abroad. It is very important to study the role of complement system in the kidney injury of TCE sensitized mice. Objective To detect the expression of C3 and C3aR in the kidney of mice sensitized to TCE by detecting the expression of C3aR and C3aRA (SB290157), a specific antagonist of C3aR. The effect of complement system on renal injury in TCE-sensitized mice was revealed, which provided a basis for exploring the mechanism of renal injury in occupational trichloroethylene drug Eruption-like dermatitis (ODMLT). After one week of sexual feeding, the experimental animals were randomly divided into blank control group (5 rats), solvent control group (5 rats), TCE treatment group (16 rats) and TCE+C3aRA combined treatment group (16 rats). When the score of the mice was more than 1, the skin sensitized mice were divided into sensitized group and non-sensitized group. The treatment group was divided into sensitized group and non-sensitized group. On the third day after the last stimulation, the eyeballs were collected and the kidney tissues were aseptically removed. The paraffin sections and frozen sections were prepared using the kidney tissues. Creatinine (CRE), urea nitrogen (BUN) detection kit, detection of serum renal function related indicators CRE, BUN levels; kidney HE staining to observe renal pathological changes; immunohistochemical detection of renal tissue related inflammatory factors expression level; real-time quantitative RT-PCR detection of renal tissue C3, C3aR and inflammatory factors reverse transcription level; The expression of podocin in kidney tissue was detected by epidemic fluorescence, and the expression of C3aR and Podocin protein in kidney tissue was detected by Western Blotting. Results (1) Sensitization: The growth of mice was normal during the experiment, and there was no significant difference in body weight among the groups. The sensitization rate was 7/16 (43.75%) in the TCE group and 6/16 (37.5%) in the TCE + C3aRA group. (2) No obvious pathological changes were found in the kidneys of the blank control group, solvent control group and all non-sensitized mice. Renal tubular epithelial cell lysis. After C3aRA treatment, inflammatory cell infiltration in the kidney of sensitized mice was significantly reduced, renal tubular injury was alleviated, and renal injury was significantly improved. (3) Renal function test results: There was no significant difference in CRE and BUN levels between the solvent control group and the non-sensitized group, and there were no significant differences in CRE and BUN levels between the TCE sensitized group and the TCE+C3aRA sensitized group. However, the levels of CRE and BUN in TCE+C3aRA sensitized group were significantly lower than those in TCE sensitized group after C3aRA treatment. (4) The results of C3aR determination in renal tissue showed that there was no significant change in transcription level of C3aR in TCE sensitized group compared with blank control group and solvent control group. The expression of C3aR protein in kidney tissue of mice sensitized with TCE was significantly higher than that in non-sensitized group. Western Blotting results also showed that the expression of C3aR protein was lower in blank control group, solvent control group and TCE non-sensitized group, but the expression of C3aR protein was significantly increased in TCE sensitized group. Compared with the blank control group and solvent control group, there was no significant change in C3 transcription level in TCE non-sensitized group, and C3 transcription level in TCE sensitized group was significantly increased. (6) Determination of cytokines IL-1beta and TNF-alpha in renal tissue: IL-1beta and TNF-alpha in renal tissue between blank control group and solvent control group and non-sensitized group. The levels of IL-1 beta and TNF-alpha in renal tissue of TCE sensitized group and TCE+C3aRA sensitized group were higher than those of solvent control group and corresponding non-sensitized group. No significant expression of IL-1beta and TNF-a was found in the blank control group and solvent control group, but in the kidney tissues of TCE sensitized group and TCE+C3aRA sensitized group, and the expression levels of IL-1beta and TNF-a were decreased in TCE+C3aRA sensitized group. (7) Determination of podocin protein in the kidney tissues of blank control group and solvent The expression of Podocin protein in kidney of mice sensitized with TCE and sensitized with TCE+C3aRA was significantly lower than that of control group and solvent control group, but the expression of Podocin protein in TCE+C3aRA sensitized group was significantly higher than that of TCE sensitized group. Conclusion (1) The expression of complement C3 and C3aR in the kidney of TCE-sensitized mice was significantly up-regulated, and there was local complement activation in the kidney of TCE-sensitized mice. (2) Complement C3aR signaling aggravated the immunity of TCE-sensitized mice by up-regulating the secretion of inflammatory factors IL-1beta and TNF-alpha. (4) Complement C3aR signal may affect the expression of Podocin protein in the kidneys of TCE-sensitized mice.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.7
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