【作者】 冷静；

【导师】 朱启星； 沈彤；

【作者基本信息】 安徽医科大学 ， 劳动卫生与环境卫生学， 2011， 硕士

【摘要】 研究背景三氯乙烯(TCE)是一种有机溶剂,在工业上作为脱脂、清洗和去污剂应用广泛。TCE职业暴露的工人出现三氯乙烯药疹样皮炎(DMLT)已经成为国内主要的职业卫生问题之一,有关TCE引起危害的案例时有报道,靶器官主要是肝脏、肾脏、神经、皮肤等。目前国内外相关研究主要在皮肤和肝脏等领域,对TCE导致肾脏损伤的研究较少,机制仍然不明。研究目的根据不同组别以及时点豚鼠肾脏中MDA含量和SOD活力以及尿液中ALB、α1-MG、β2-MG、γ-GGT和AAP含量的变化,结合肾功能和病理学检查,探讨TCE对肾脏的损伤作用。研究方法选用体重250～300g/只,白色雌性豚鼠,经适应性饲养一周后,随机分成空白对照组、溶剂对照组(橄榄油)、TCE暴露组和2,4-二硝基氯苯(DNCB)对照组。采用豚鼠最大值试验(Guinea Pig Maximization Test, GPMT)方法建立豚鼠致敏模型,观察并记录豚鼠背部皮肤反应情况并进行拍照;依据《化学品毒性鉴定技术规范》(卫监督发【2005】272号)中规定的皮肤反应评分标准对豚鼠皮肤反应进行评分;当动物皮肤反应积分≥1时,为皮肤致敏反应阳性,该动物被划分为致敏组,反之被划分为未致敏组。分别在末次激发前一天和末次激发后24h、72h分三批收集豚鼠24小时尿液,采用ELISA方法测定尿蛋白及尿酶等相关指标,抽取豚鼠血液制备血清,用于测肾功能;处死动物,无菌取肾脏,用甲醛及戊二醛固定,分别用于HE染色及透射电子显微镜观察病理;组织匀浆后测定MDA和SOD变化。用SPSS15.0统计软件包对实验结果进行统计分析。结果1.致敏率在皮肤致敏反应试验中,空白组、溶剂对照组及TCE14d组未见红斑或水肿,在TCE试验组的26只动物中有17只出现红斑水肿,致敏率为65.38%,DNCB实验组全部出现红斑或水肿。2.病理学检查结果HE染色和超微病理结果显示,TCE致敏组肾小球毛细血管基底膜节段增厚或者菲薄,足突部分融合,肾小管上皮细胞线粒体肿胀、空泡变性,微绒毛萎缩,个别小管呈萎缩状。3.肾功能检查结果溶剂对照组肾功能各指标与空白组相比,差异无统计学意义(p>0.05);与溶剂对照组及TCE14d组相比,TCE致敏组两个时点血清BUN水平均明显升高(P<0.05);TCE致敏组血清BUN水平均较非致敏组高(P<0.05);与相应时点DNCB对照组相比,TCE致敏72h组升高(P<0.05)。与溶剂对照组相比,TCE致敏组两个时点血清Cr水平有升高趋势,但差异无统计学意义(P>0.05);TCE致敏24h组高于未致敏24h组;与相应时点的DNCB相比,TCE致敏组血清Cr水平均较高(P<0.05)。4. mALB检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比,TCE14d组、TCE致敏组及未致敏组豚鼠24小时尿液中mALB含量均明显升高(p<0.05);与TCE14d组和TCE未致敏组相比,TCE致敏组尿液中mALB含量均明显升高(p<0.05);与相应时点的DNCB对照组相比,TCE致敏组和未致敏组mALB含量均明显升高(p<0.05)。5.α1-MG检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比, TCE致敏组、未致敏组各时点均明显升高,且差异有统计学意义(p<0.05);与TCE14d组相比,TCE致敏组和未致敏组豚鼠尿液中α1-MG含量均明显升高(p<0.05);与TCE未致敏组相比,TCE致敏72h组豚鼠尿液中α1-MG含量明显升高(p<0.05);与相应时点的DNCB对照组相比,TCE致敏组豚鼠24小时尿液中α1-MG含量明显升高(p<0.05)。6.β2-MG检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比,TCE致敏组各时点均明显升高(p<0.05);与TCE14d组相比,TCE致敏组尿液中β2-MG含量均明显升高(p<0.05);与TCE未致敏组相比,TCE致敏24h组豚鼠尿液中β2-MG含量明显升高(p<0.05);与相应时点的DNCB对照组相比,TCE致敏组豚鼠尿液中β2-MG含量明显升高(p<0.05)。7. AAP检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比,TCE致敏组各时点均明显升高(p<0.05);与TCE14d组相比,TCE致敏组24h尿液中AAP活性明显升高(p<0.05);与TCE未致敏组相比,TCE致敏组豚鼠尿液中AAP活性均明显升高(p<0.05);与相应时点的DNCB对照组相比,TCE致敏组豚鼠尿液中AAP活性均明显升高(p<0.05)。8. Y-GGT检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比,TCE14d组、TCE致敏组各时点均明显升高(p<0.05);与TCE未致敏组相比,TCE致敏24h、72h组豚鼠尿液中γ-GGT活性稍高,但差异无统计学意义(p>0.05);与相应时点的DNCB对照组相比,TCE致敏组豚鼠尿液中γ-GGT活性均明显升高(p<0.05)。9. MDA和SOD检测结果溶剂对照组与空白组相比差异无统计学意义(p>0.05);与溶剂对照组相比,TCE致敏24h组MDA含量明显上升(P<0.05);TCE致敏组与未致敏组比较,24h组MDA含量差异有统计学意义(P<0.05)。TCE致敏组SOD活性比溶剂对照组明显降低(P<0.05);TCE致敏24h、72h组SOD活性较非致敏组低,差异有统计学意义(P<0.05)。结论1、采用豚鼠最大值试验可以建立TCE致敏模型,本实验条件下,TCE致敏率为65.38%。2、TCE暴露可造成豚鼠肾脏的病理性和功能性损伤;TCE对豚鼠肾脏的损害不局限于肾小球,还累及肾小管,且对肾小管的损害程度重于肾小球。3、尿蛋白和尿酶可能作为影响因素和氧化应激一起在TCE致敏豚鼠肾脏损伤中发挥重要作用。4、TCE对肾脏的作用机制不同于DNCB,TCE不单纯是致敏剂,也是免疫损伤剂。更多还原

【Abstract】 Background Trichloroethylene (TCE) is an organic solvent mainly used as degreaser in industry. Occupational exposing to TCE, workers appear dermatitis (DMLT) with poly-organ injury, and it has become a major occupational health issue now. In recent years, there are many studies on the mechanism of skin and liver injury, but there are little studies on the mechanism of renal injuryObjective Detecting MDA, SOD, ALB,α1-MG,β2-MG,γ-GGT and AAP expression of Guinea Pigs in different groups and different time points, combined with renal function tests and pathological examination, to investigate the mechanism of renal injury induced by TCE.Method Female guinea pigs, which were white and weighing 250～300g ,were adopted. After adaptive feeding a week, the Guinea Pigs were divided to blank control group, solvent (olive oil) control group, DNCB control grouTCE group in random. To monitor the status of guinea-pig growth and health, their weight was measured regularly. Adopting Guinea Pig Maximization Test (GPMT) to establish sensitization model, to observe and record the skin reaction in Guinea Pigs back and take pictures. Reaction in Guinea Pigs skin was scored in accordance with the skin reaction score standards in“Toxic chemicals identified technical specifications”(the Ministry of Health superintend made【2005】No.272). When the skin reaction scores of test group≥1, it should be ruled positive skin sensitization, which was sensitized group, otherwise was non-sensitized group. 24h before, 24h and 72h after sensitization test and collected total volume urine of Guinea Pigs,suctioned the serum and killed the animals. Serum was prepared for renal function. Urine protein and urease were detected by ELISA. MDA and SOD in renal homogenate were determined by chemistry. Experimental results were analyzed with SPSS15.0.Result1. Sensitization rate In the skin sensitization test, the control group, solvent control group showed no erythema or edema; 17 of 26 animals in TCE test group showed erythema and edema, sensitization rate is 65.38%.All animals in DNCB control group showed erythema and edema.2. Pathological test results The kidneys of the normal control group exhibited normal cortical morphology as determined by light microscope and electron microscope.By HE staining and electron microscope, TCE sensitized group was observed segmental glomerular basement membrane thickening or meager, some fusion of foot processes, tubular epithelial cell mitochondrial swelling, vacuolar degeneration and atrophy of microvilli, individual tubules were Atrophy-like.3. Results of liver function Compared with the control group, renal function of solvent group was not statistically significant (p>0.05); Compared with the solvent control group and TCE non-provocated group,BUN level in TCE sensitized group elevated significantly (p<0.05); Compared with TCE non-sensitized group, BUN level in TCE sensitized 24h and 72h group increased significantly(p<0.05) ; Compared with DNCB control group,BUN levels in TCE sensitized 24h group increased significantly(p<0.05) . Compared with the solvent control group, Cr level in TCE sensitized group showed no significant difference(p>0.05),but increased significantly compared with the DNCB control group.4. Results of mALB Compared with the blank control group, mALB level in solvent control group showed no significant difference(p>0.05); Compared with the solvent control group, mALB in TCE exposured group elevated significantly(p<0.05); mALB were significantly increased in TCE sensitized group compared with TCE non-sensitized group or TCE non-provocated group (p<0.05);Compared with DNCB control group,mALB in TCE sensitized and non-sensitized group both increased significantly (p<0.05).5. Results ofα1-MG Compared with the blank control group,α1-MG levels in solvent control group showed no significant difference (p>0.05); Compared with the solvent control group or TCE non-provocated group,α1-MG levels in TCE sensitized and non-sensitized group both increased significantly (p<0.05); Compared with TCE non-sensitized group,α1-MG levels in TCE sensitized 72h group increased significantly (p<0.05);α1-MG levels were significantly increased in TCE sensitized 24h group compared with DNCB control group (p<0.05).6. Results ofβ2-MG Compared with the blank control group,β2-MG levels in solvent control group showed no significant difference (p>0.05); Compared with the solvent control group,TCE non-provocated group or DNCB control group,β2-MG levels in TCE sensitized group increased significantly (p<0.05); Compared with TCE non-sensitized group,β2-MG levels in TCE sensitized 24h increased significantly(p<0.05).7. Results of AAP Compared with the blank control group, AAP levels in solvent control group showed no significant difference (p>0.05); Compared with the solvent control group, DNCB control group or TCE non-sensitized group, AAP levels in TCE sensitized group increased significantly (p<0.05); Compared with TCE non-provocated group, AAP levels in TCE sensitized 24h group increased significantly(p<0.05).8. Results of Y-GGT Compared with the blank control group, Y-GGT levels in solvent control group showed no significant difference (p>0.05); Compared with the solvent control group, Y-GGT levels in TCE non- provocated group and TCE sensitized group increased significantly (p<0.05); Y-GGT were significantly increased in TCE sensitized group compared with DNCB control group (p<0.05).9. MDA and SOD The content of MDA in kidney of the TCE sensitized groups were increased (p<0.05) at the point of 24h, while T-SOD activity decreased significantly (p<0.05) at the all time. At the point of 24h, the content of MDA in kidney of the TCE sensitized groups were higher than those in no-sensitized groups (P<0.05). Compared with no-sensitized groups, the T-SOD activity decreased significantly at the point of 24h and 72h.Conclusion1. TCE sensitized model was established by Guinea Gigs Maximum Test, TCE sensitization rate was 65.38%.2. TCE can cause renal pathological damage and renal function injury; TCE-induced kidney damage in guinea pigs are not limited to glomeruli, but also involving the renal tubules, and the renal tubular damage was severe in the glomeruli. 3. Urinary protein and urinary enzymes may be used as independent factors,combined with oxidative stress,may have participated in renal injury induced by TCE.4. In the mechanisms of renal injury,TCE is different from DNCB;TCE is not only a simple allergic agent, but also is immune damage agent.更多还原