【摘要】：研究背景三氯乙烯(Trichloroethylene,TCE),作為一種常見金屬洗滌劑、脂類萃取劑、涂料稀釋劑廣泛應(yīng)用于電子、電鍍、制藥等行業(yè)。TCE可以引起職業(yè)接觸人群發(fā)生職業(yè)性三氯乙烯藥疹樣皮炎(Occupational Dermatitis Medicamentosa-like of Trichloroethylene,ODMLT)。ODMLT可出現(xiàn)多系統(tǒng)、多臟器的損傷,其中部分患者可出現(xiàn)嚴重的腎臟損傷,嚴重威脅相關(guān)職業(yè)人群健康狀況。對于ODMLT發(fā)病機制,目前研究發(fā)現(xiàn)除了遲發(fā)型IV型變態(tài)反應(yīng)以外,補體系統(tǒng)在發(fā)病過程中可能也起到了重要的作用。在前期的動物實驗研究中發(fā)現(xiàn)腎臟損傷中存在著補體系統(tǒng)的激活,補體C3a在致敏小鼠腎臟組織中有明顯的沉積現(xiàn)象。C3a可與其特異性受體C3aR相結(jié)合,發(fā)揮擴張血管、趨化炎癥細胞、調(diào)節(jié)炎癥反應(yīng)等功能。國內(nèi)外研究發(fā)現(xiàn)C3a-C3aR系統(tǒng)在多種腎臟損傷中起到了十分重要作用,故深入研究補體系統(tǒng)在TCE致敏小鼠腎臟損傷中的作用對探討ODMLT發(fā)病機制有著十分積極的意義。研究目的本研究通過檢測小鼠腎臟中C3及C3aR的表達,使用C3aR特異性拮抗劑C3aRA(SB290157),來檢測其對TCE致敏小鼠腎臟損傷的影響,揭示補體系統(tǒng)在TCE致敏小鼠免疫性腎損傷中的作用,進一步為探索職業(yè)性三氯乙烯藥疹樣皮炎(ODMLT)腎臟損傷機制提供依據(jù)。研究方法選取42只無特定病原體(SPF)級,雌性6-8周BALB/c小鼠,體重范圍17.1-24.7(20.3±1.7)g。在適應(yīng)性喂養(yǎng)一周后,根據(jù)隨機分配原則將實驗動物隨機分為:空白對照組(5只)、溶劑對照組(5只)、TCE處理組(16只)和TCE+C3aRA聯(lián)合處理組(16只)。參照BALB/c小鼠三氯乙烯致敏模型方法,在末次激發(fā)24h后,根據(jù)小鼠皮膚反應(yīng)狀況,按照皮膚致敏試驗評分標準進行分組,當小鼠積分≥1分時,認為皮膚致敏陽性即分為致敏組,否則即判定為非致敏組,以此將處理組區(qū)分為致敏組和非致敏組。予末次激發(fā)后第3天,眼球采血及無菌取出腎臟組織,使用腎臟組織制備石蠟切片和冰凍切片。采用肌酐(CRE)、尿素氮(BUN)檢測試劑盒,檢測血清中腎功能相關(guān)指標CRE、BUN的水平;腎臟HE染色來觀察腎臟病理變化狀況;免疫組化檢測腎臟組織中相關(guān)炎癥因子的表達水平;實時熒光定量RT-PCR檢測腎臟組織中C3、C3aR及炎癥因子的逆轉(zhuǎn)錄水平;免疫熒光檢測腎臟組織中足細胞蛋白Podocin的表達;Western Blotting檢測腎臟組織中C3aR、Podocin蛋白的表達。研究結(jié)果(1)致敏情況:試驗期間小鼠生長正常,各組間小鼠體重改變無明顯差異�？瞻讓φ战M和溶劑對照組小鼠皮膚未見明顯變化,根據(jù)皮膚反應(yīng)情況,TCE處理組致敏率7/16(43.75%),TCE+C3aRA處理組致敏率6/16(37.5%)。(2)腎臟病理結(jié)果顯示:在空白對照組、溶劑對照組和所有非致敏組小鼠腎臟中未發(fā)現(xiàn)明顯的病理改變。TCE致敏組腎臟中可見明顯的炎性細胞浸潤,腎小管結(jié)構(gòu)改變,腎小管上皮細胞溶解現(xiàn)象。在C3aRA處理后致敏小鼠腎臟炎性細胞浸潤明顯減少、腎小管損傷減輕,腎臟損傷得到明顯改善。(3)腎功能檢測結(jié)果:溶劑對照組與各非致敏組之間CRE、BUN水平均未發(fā)現(xiàn)明顯差異,TCE致敏組和TCE+C3aRA致敏組CRE、BUN均高于溶劑對照組和相應(yīng)的非致敏組。但是,發(fā)現(xiàn)在C3aRA處理后,TCE+C3aRA致敏組CRE、BUN水平均明顯低于TCE致敏組。(4)腎臟組織中C3aR測定結(jié)果:與空白對照組和溶劑對照組相比,TCE非致敏組C3aR在轉(zhuǎn)錄水平上均未見明顯變化。與溶劑對照組和相應(yīng)未致敏組比較,TCE致敏組小鼠腎臟組織中C3aR在轉(zhuǎn)錄水平上顯著上升。Western Blotting結(jié)果也顯示,空白對照組和溶劑對照組以及TCE非致敏組中C3aR蛋白的表達量較低,但是在TCE致敏組中C3aR蛋白的表達明顯上升。(5)腎臟組織中C3測定結(jié)果:與空白對照組和溶劑對照組相比,TCE非致敏組在C3轉(zhuǎn)錄水平上未見明顯變化,TCE致敏組腎臟組織中C3在轉(zhuǎn)錄水平上顯著上升。(6)腎臟組織中細胞因子IL-1β、TNF-α測定結(jié)果:空白對照組和溶劑對照組與各非致敏組之間腎臟組織中IL-1β、TNF-α轉(zhuǎn)錄水平均未發(fā)現(xiàn)明顯變化,TCE致敏組和TCE+C3aRA致敏組腎臟組織中IL-1β、TNF-α均高于溶劑對照組和相應(yīng)非致敏組。但是發(fā)現(xiàn)在C3aRA處理后,TCE+C3aRA致敏組腎臟組織中IL-1β、TNF-α轉(zhuǎn)錄水平明顯低于TCE致敏組。免疫組化也呈現(xiàn)相同的結(jié)果,在空白對照組和溶劑對照組中均未發(fā)現(xiàn)有IL-1β、TNF-α的明顯表達,而在TCE致敏組和TCE+C3aRA致敏組腎臟組織中有明顯的表達,且TCE+C3aRA致敏組IL-1β、TNF-α較的表達水平有所下降。(7)腎臟組織中足細胞Podocin蛋白測定結(jié)果:在空白對照組和溶劑對照組中Podocin蛋白可見正常表達,與空白對照組和溶劑對照組相比TCE致敏組和TCE+C3aRA致敏組小鼠腎臟Podocin蛋白的表達明顯降低,但是與TCE致敏組相比,TCE+C3aRA致敏組Podocin蛋白表達水平有明顯回升。Western Blotting結(jié)果也顯示,與TCE致敏組相比,TCE+C3aRA致敏組與Podocin蛋白表達水平有明顯回升。結(jié)論(1)TCE致敏小鼠腎臟中補體C3和C3aR表達明顯上調(diào),TCE致敏小鼠腎臟損傷中存在局部補體激活的現(xiàn)象。(2)補體C3aR信號通過上調(diào)炎癥因子IL-1β和TNF-α的分泌,加重了TCE致敏小鼠免疫性腎臟損傷(3)TCE致敏小鼠免疫性腎損傷中Podocin蛋白的表達下調(diào)。(4)補體C3aR信號可能影響了TCE致敏小鼠腎臟中Podocin蛋白表達。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R135.7

【相似文獻】

相關(guān)期刊論文 前2條

1 李偉真;董元寶;高翔;劉如參;;尾靜脈注射C3a高表達腺病毒對自發(fā)性免疫性心肌炎的影響[J];中國現(xiàn)代藥物應(yīng)用;2012年03期

2 吳青松;李治國;劉廣波;高毅;;人源肝細胞系CL-1、HepFMMU、HepG2、C3A生物學(xué)特性與功能的比較[J];實用醫(yī)學(xué)雜志;2012年04期

相關(guān)會議論文 前1條

1 溫勇;周偉玲;劉加平;;C_3A早期水化Zeta電位的研究[A];第九屆全國水泥和混凝土化學(xué)及應(yīng)用技術(shù)會議論文匯編（上卷）[C];2005年

，

本文編號：2223606