血浆激肽释放酶激肽系统在三氯乙烯致敏小鼠肾脏损伤中的作用研究

发布时间：2018-05-14 14:45

  本文选题：三氯乙烯 + BALB/c小鼠　； 参考：《安徽医科大学》2015年硕士论文

【摘要】：研究背景三氯乙烯(Trichloroethylene,TCE)是一种具有挥发性的有机溶剂。由于其高效的去污能力,被广泛的用于工业中金属、线路板等物质的清洗。近年来,由于TCE大规模的生产和应用,已成为一种普遍存在的环境污染物,,长期暴露的工人还可导致职业性TCE药疹样皮炎(ODMLT)。大量的临床病例显示,肾脏损伤最后导致的肾衰竭是许多患者死亡的原因之一,故肾脏损伤越来越受到研究者的重视。有研究发现血浆激肽释放酶-激肽系统(Plasma kallikrein-kinin system,KKS)与机体的炎症损伤和免疫系统有密切关系。本课题组前期研究发现TCE致敏小鼠的肾脏中不仅有炎症性损伤,免疫性损伤也参与其中。故本研究探讨KKS在TCE致敏小鼠的免疫性肾损伤机制中的作用。研究目的参考豚鼠最大值实验以及本课题组前期的研究报道建立TCE致敏的BALB/c小鼠模型。通过腹腔注射血浆激肽释放酶的特异性抑制剂PKSI以及检测KKS中的主要成分BK、PK、B1R、B2R以及C5b-9的表达以探讨KKS在TCE致敏小鼠体内的活化情况以及其在免疫性肾损伤中的作用。研究方法选购6~8W,体重20-25g的健康雌性BALB/c小鼠。经过一周的适应性饲养后,小鼠被随机分成4组,分别是:空白对照组、溶剂对照组、TCE处理组以及PKSI处理组。参考豚鼠最大值实验以及本课题组前期的研究报道建立BALB/c小鼠致敏模型,并在两次激发前24h腹腔注射PKSI。根据小鼠背部受试部位的致敏情况以及处死的时间不同将其分为tce致敏24h、48h、72h和7d组、tce未致敏24h、48h、72h和7d组、pksi致敏24h、48h、72h和7d组和pksi未致敏24h、48h、72h和7d组。采用elisa法检测血浆中bk的表达水平。免疫荧光法检测肾脏组织中的bk、pk、b1r和b2r的沉积情况。qrt-pcr法检测肾脏组织中b1r和b2rmrna表达水平。westernblot法检测肾脏组织中b1r和b2r蛋白表达水平。免疫组化法检测c5b-9在肾脏组织中的沉积情况。结果1.致敏率本研究中的空白对照组和溶剂对照组均未见到红肿现象;tce处理组共有120只小鼠,其中有48只小鼠背部皮肤有明显的红肿,故致敏率为40%,致敏强度为中度;pksi处理组也有120只小鼠,其中仅27只小鼠背部皮肤有明显的红肿,故致敏率为22.5%,致敏强度为轻度;本研究中,总的致敏率为31.25%。2.肾脏病理检查结果通过肾脏组织的he染色结果,发现tce致敏组的肾脏中有大量的炎细胞浸润以及肾小管上皮细胞空泡样变性,而相应时点的pksi致敏组的肾脏,损伤明显减轻。其余组未见明显异常。3.肾功能检测结果肾功能检查主要对血清中bun和cr这两个具有代表性的指标进行检测。结果发现,空白对照组与溶剂对照组的bun和cr相比,差异均无统计学意义(p0.05);与溶剂对照组相比,tce致敏组的24h、48h和72h组的bun和cr表达明显增高且差异有统计学意义(p0.05);其中,tce致敏组的48h和72h组的bun表达明显高于相应时点的tce未致敏组以及pksi致敏组(p0.05)。tce致敏组的24h、48h和72h组cr的表达明显高于相应时点的未致敏组,且其72h组的表达与相应时点的pksi致敏组相比,表达明显增高,差异有统计学意义(p0.05)。4.血浆中bk的表达水平结果显示:空白对照组与溶剂对照组相比较,差异无统计学意义(p0.05)。与溶剂对照组相比,tce致敏组的四个时点以及pksi致敏组的24h和48h组的bk表达水平明显增加,且差异有统计学意义(p0.05)。tce致敏组的24h、48h和72h组与相应的未致敏组相比,bk表达水平同样显著增加,且差异有统计学意义(p0.05)。pksi致敏组的72h组与相应时点的tce致敏组相比,bk表达水平明显下降,且差异有统计学意义(p0.05)。5.肾脏组织中bk、pk、b1r和b2r的沉积情况结果显示如下:肾脏组织中bk、pk、b1r和b2r沉积的变化趋势大致相似:在空白对照组与溶剂对照组中的沉积都比较少,甚至没有;而在tce致敏组中四个指标的沉积量随着时点的变化逐渐增加,并在72h时点达到最高峰,在7d时有所恢复;pksi致敏组的四个时点的四个指标都较tce致敏组的相应时点的沉积量相对降低。6.肾脏组织中b1r和b2rmrna表达情况采用qrt-pcr法检测肾脏组织中b1r和b2rmrna的表达水平。结果如下:空白对照组与溶剂对照组相比,两个指标的差异均无统计学意义(p0.05)。tce致敏组的四个时点以及pksi致敏组的24h和48h组的b1rmrna表达水平与溶剂对照组相比较明显增加,且差异有统计学意义(p0.05)。tce致敏组的b2rmrna表达水平也是逐渐增加的,且在72h时点达到最高。pksi致敏组的b1r以及b2rmrna表达水平都较tce致敏组的相应时点相对降低。在72h时点组,tce致敏组比pksi致敏组的b1r和b2rmrna表达水平显著增加,且差异有统计学意义(p0.05)。7.肾脏组织中b1r和b2r的蛋白表达水平采用westernblot法检测肾脏组织中b1r以及b2r的蛋白表达水平。结果显示:空白对照组与溶剂对照组的b1r蛋白几乎不表达,b2r的表达也比较少。tce致敏组的b1r和b2r的表达明显比溶剂对照组增加,且差异有统计学意义(p0.05)。两个受体在tce致敏组中的表达也明显比pksi致敏组多,差异有统计学意义(p0.05)。8.肾脏组织中c5b-9的沉积情况结果显示如下:在空白对照组与溶剂对照组中c5b-9的表达都比较少,且差异无统计学意义(p0.05);在tce致敏组中24h、48h、72h沉积量逐渐增加,并在72h组达到最高峰,与溶剂对照组相比显著升高,差异有统计学意义(p0.05),在7d时有所降低;pksi致敏组的四个时点的c5b-9表达含量都较TCE致敏组的相应时点的表达明显降低,且在48h和72h时点两组差异有统计学意义(P0.05)。结论1.一定剂量的TCE可引起小鼠皮肤致敏反应并可导致小鼠的免疫性肾损伤。2.TCE致敏小鼠体内补体激活的过程中还伴有KKS的活化,且KKS的活化及其产物在TCE所致的免疫性肾损伤过程中可能起重要作用。
[Abstract]:Trichloroethylene (TCE) is a kind of volatile organic solvent. Because of its efficient decontamination ability, it is widely used in the cleaning of metal, circuit board and other materials in industry. In recent years, because of the large-scale production and application of TCE, it has become a common environmental pollutant, and the workers who have been exposed for a long time have also been used. It can lead to occupational TCE rash like dermatitis (ODMLT). A large number of clinical cases show that renal failure resulting from renal injury is one of the causes of death in many patients. Therefore, renal injury is becoming more and more important for the researchers. Studies have found that plasma kinin releasing enzyme - kinin system (Plasma kallikrein-kinin system, KKS) and inflammation of the body have been found. Injury is closely related to the immune system. In our previous study, we found that there were not only inflammatory damage and immune injury in the kidneys of TCE sensitized mice, but also the role of KKS in the mechanism of immune renal injury in TCE sensitized mice. This paper reports the establishment of a TCE sensitized BALB/c mouse model. By intraperitoneal injection of the specific inhibitor PKSI of plasma kinin releasing enzyme and the expression of the main components of KKS, BK, PK, B1R, B2R and C5b-9, the activation of KKS in TCE sensitized mice and its role in immune renal injury are investigated. 20-25g healthy female BALB/c mice were randomly divided into 4 groups after a week of adaptation. The mice were randomly divided into 4 groups: blank control group, solvent control group, TCE treatment group and PKSI treatment group. The BALB/ c mice sensitization model was established by reference to the guinea pig maximum experiment and the previous research report of this group, and the 24h intraperitoneal injection before two times of stimulation. PKSI. was divided into TCE sensitized 24h, 48h, 72h and 7d group, TCE without sensitizing 24h, 48h, 72h and 7d groups, pksi sensitized, pksi and non sensitized groups. The deposition of BK, PK, B1R and B2R in the renal tissue, B1R and b2rmrna expression levels were detected by.Qrt-pcr method and.Westernblot method was used to detect the expression level of B1R and B2R protein in renal tissue. Immunohistochemical method was used to detect the deposition of C5b-9 in renal tissue. Results the 1. sensitization rate in the blank control group and the solvent control group did not see red. There were 120 mice in the TCE treatment group, of which 48 mice had obvious red and swollen back skin, the sensitization rate was 40%, the sensitization intensity was moderate, and the pksi treatment group also had 120 mice, of which only 27 mice had obvious red and swollen back skin, and the sensitization rate was 22.5% and the sensitization intensity was mild. The total sensitization rate was 31.25%.2. in this study. The renal pathological examination results of renal tissue HE staining results showed that there were a large number of inflammatory cells infiltrating in the kidneys of the TCE sensitizing group and the vacuolated degeneration of renal tubular epithelial cells, while the kidney in the pksi sensitizing group at the corresponding time point was significantly reduced. No significant abnormal.3. renal function test results were not found in the other groups. The two representative indexes of bun and Cr in the Qing Dynasty were detected. The results showed that there was no significant difference between the blank control group and the bun and Cr in the solvent control group (P0.05). Compared with the solvent control group, the bun and Cr expressions of the 24h, 48h and 72h groups in the TCE sensitizing group were significantly higher and the difference was statistically significant (P0.05). The expression of BUN in the 48h and 72h groups was significantly higher than that in the TCE non sensitization group at the corresponding time point and the 24h in the pksi sensitizing group (P0.05).Tce sensitizing group. The expression of Cr in 48h and 72h groups was significantly higher than that in the non sensitized group at the corresponding time points, and the expression of the 72h group was significantly higher than that in the corresponding time point sensitizing group. The expression level of BK showed that there was no significant difference between the blank control group and the solvent control group (P0.05). Compared with the solvent control group, the four time points of the TCE sensitizing group and the BK expression level of the 24h and 48h groups in the pksi sensitizing group were significantly increased, and the difference was statistically significant (P0.05).Tce sensitizing group 24h, 48h and 72h groups and corresponding groups. The expression level of BK was also significantly increased in the non sensitized group, and the difference was statistically significant (P0.05) in the 72h group of.Pksi sensitizing group, the BK expression level decreased significantly compared with the TCE sensitizing group at the corresponding time points, and the difference was statistically significant (P0.05).5. in the renal tissue, BK, PK, B1R, and B2R. The change trend of the B2R deposition is roughly similar to that in the blank control group and the solvent control group, and the deposition of the four indexes in the TCE sensitizing group gradually increases with the time point, reaching the peak at the 72h point and recovering at the 7d, and the four indexes of the four time points of the pksi sensitizing group are all TCE The deposition of the corresponding time points in the sensitized group decreased the expression of B1R and b2rmrna in.6. kidney tissue. The expression of B1R and b2rmrna in renal tissue was detected by qRT-PCR method. The results were as follows: the difference between the two indexes of the blank control group and the solvent control group was not statistically meaning sense (P0.05).Tce sensitizing group at four time points and pksi. The expression level of b1rmrna in the 24h and 48h groups of the sensitized group was significantly higher than that in the solvent control group, and the difference was statistically significant (P0.05) in the.Tce sensitized group, the b2rmrna expression level was gradually increased, and the B1R and b2rmrna expression of the highest.Pksi sensitized group at the time point of 72h were relatively lower than the corresponding time points of the TCE sensitizing group. 7 2H time point group, the expression level of B1R and b2rmrna in TCE sensitizing group was significantly higher than that of pksi sensitized group, and the difference was statistically significant (P0.05) the protein expression level of B1R and B2R in.7. kidney tissue was detected by Westernblot method for B1R and B2R protein expression in renal tissue. The expression of B2R and the expression of B1R and B2R in the.Tce sensitized group was significantly higher than that in the solvent control group, and the difference was statistically significant (P0.05). The expression of two receptors in the TCE sensitized group was significantly more than that in the pksi sensitized group, and the difference was statistically significant (P0.05) the deposition of C5b-9 in the.8. renal tissue showed as follows: in the empty space The expression of C5b-9 in the white control group and the solvent control group was less, and the difference was not statistically significant (P0.05). In the TCE sensitizing group, the deposition of 24h, 48h, 72h increased gradually and reached the peak in the 72h group. The difference was significantly higher than that in the solvent control group. The difference was statistically significant (P0.05) and decreased at 7d; the four time points of the pksi sensitization group were c5b-. The expression of 9 was significantly lower than that at the corresponding time points in the TCE sensitized group, and there was a significant difference between the two groups at the time point of 48h and 72h (P0.05). Conclusion 1. a certain dose of TCE could cause the skin sensitization of mice and could lead to the activation of the complement activation of the complement in the sensitized mice induced by the immune renal injury in.2.TCE, and K Activation of KS and its products may play an important role in TCE induced immune renal injury.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R135.7

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