本文選題：三氯乙烯 + BALB/c小鼠�。� 參考：《安徽醫(yī)科大學(xué)》2015年碩士論文

【摘要】：研究背景三氯乙烯(Trichloroethylene,TCE)是一種具有揮發(fā)性的有機溶劑。由于其高效的去污能力,被廣泛的用于工業(yè)中金屬、線路板等物質(zhì)的清洗。近年來,由于TCE大規(guī)模的生產(chǎn)和應(yīng)用,已成為一種普遍存在的環(huán)境污染物,,長期暴露的工人還可導(dǎo)致職業(yè)性TCE藥疹樣皮炎(ODMLT)。大量的臨床病例顯示,腎臟損傷最后導(dǎo)致的腎衰竭是許多患者死亡的原因之一,故腎臟損傷越來越受到研究者的重視。有研究發(fā)現(xiàn)血漿激肽釋放酶-激肽系統(tǒng)(Plasma kallikrein-kinin system,KKS)與機體的炎癥損傷和免疫系統(tǒng)有密切關(guān)系。本課題組前期研究發(fā)現(xiàn)TCE致敏小鼠的腎臟中不僅有炎癥性損傷,免疫性損傷也參與其中。故本研究探討KKS在TCE致敏小鼠的免疫性腎損傷機制中的作用。研究目的參考豚鼠最大值實驗以及本課題組前期的研究報道建立TCE致敏的BALB/c小鼠模型。通過腹腔注射血漿激肽釋放酶的特異性抑制劑PKSI以及檢測KKS中的主要成分BK、PK、B1R、B2R以及C5b-9的表達以探討KKS在TCE致敏小鼠體內(nèi)的活化情況以及其在免疫性腎損傷中的作用。研究方法選購6~8W,體重20-25g的健康雌性BALB/c小鼠。經(jīng)過一周的適應(yīng)性飼養(yǎng)后,小鼠被隨機分成4組,分別是:空白對照組、溶劑對照組、TCE處理組以及PKSI處理組。參考豚鼠最大值實驗以及本課題組前期的研究報道建立BALB/c小鼠致敏模型,并在兩次激發(fā)前24h腹腔注射PKSI。根據(jù)小鼠背部受試部位的致敏情況以及處死的時間不同將其分為tce致敏24h、48h、72h和7d組、tce未致敏24h、48h、72h和7d組、pksi致敏24h、48h、72h和7d組和pksi未致敏24h、48h、72h和7d組。采用elisa法檢測血漿中bk的表達水平。免疫熒光法檢測腎臟組織中的bk、pk、b1r和b2r的沉積情況。qrt-pcr法檢測腎臟組織中b1r和b2rmrna表達水平。westernblot法檢測腎臟組織中b1r和b2r蛋白表達水平。免疫組化法檢測c5b-9在腎臟組織中的沉積情況。結(jié)果1.致敏率本研究中的空白對照組和溶劑對照組均未見到紅腫現(xiàn)象;tce處理組共有120只小鼠,其中有48只小鼠背部皮膚有明顯的紅腫,故致敏率為40%,致敏強度為中度;pksi處理組也有120只小鼠,其中僅27只小鼠背部皮膚有明顯的紅腫,故致敏率為22.5%,致敏強度為輕度;本研究中,總的致敏率為31.25%。2.腎臟病理檢查結(jié)果通過腎臟組織的he染色結(jié)果,發(fā)現(xiàn)tce致敏組的腎臟中有大量的炎細胞浸潤以及腎小管上皮細胞空泡樣變性,而相應(yīng)時點的pksi致敏組的腎臟,損傷明顯減輕。其余組未見明顯異常。3.腎功能檢測結(jié)果腎功能檢查主要對血清中bun和cr這兩個具有代表性的指標(biāo)進行檢測。結(jié)果發(fā)現(xiàn),空白對照組與溶劑對照組的bun和cr相比,差異均無統(tǒng)計學(xué)意義(p0.05);與溶劑對照組相比,tce致敏組的24h、48h和72h組的bun和cr表達明顯增高且差異有統(tǒng)計學(xué)意義(p0.05);其中,tce致敏組的48h和72h組的bun表達明顯高于相應(yīng)時點的tce未致敏組以及pksi致敏組(p0.05)。tce致敏組的24h、48h和72h組cr的表達明顯高于相應(yīng)時點的未致敏組,且其72h組的表達與相應(yīng)時點的pksi致敏組相比,表達明顯增高,差異有統(tǒng)計學(xué)意義(p0.05)。4.血漿中bk的表達水平結(jié)果顯示:空白對照組與溶劑對照組相比較,差異無統(tǒng)計學(xué)意義(p0.05)。與溶劑對照組相比,tce致敏組的四個時點以及pksi致敏組的24h和48h組的bk表達水平明顯增加,且差異有統(tǒng)計學(xué)意義(p0.05)。tce致敏組的24h、48h和72h組與相應(yīng)的未致敏組相比,bk表達水平同樣顯著增加,且差異有統(tǒng)計學(xué)意義(p0.05)。pksi致敏組的72h組與相應(yīng)時點的tce致敏組相比,bk表達水平明顯下降,且差異有統(tǒng)計學(xué)意義(p0.05)。5.腎臟組織中bk、pk、b1r和b2r的沉積情況結(jié)果顯示如下:腎臟組織中bk、pk、b1r和b2r沉積的變化趨勢大致相似:在空白對照組與溶劑對照組中的沉積都比較少,甚至沒有;而在tce致敏組中四個指標(biāo)的沉積量隨著時點的變化逐漸增加,并在72h時點達到最高峰,在7d時有所恢復(fù);pksi致敏組的四個時點的四個指標(biāo)都較tce致敏組的相應(yīng)時點的沉積量相對降低。6.腎臟組織中b1r和b2rmrna表達情況采用qrt-pcr法檢測腎臟組織中b1r和b2rmrna的表達水平。結(jié)果如下:空白對照組與溶劑對照組相比,兩個指標(biāo)的差異均無統(tǒng)計學(xué)意義(p0.05)。tce致敏組的四個時點以及pksi致敏組的24h和48h組的b1rmrna表達水平與溶劑對照組相比較明顯增加,且差異有統(tǒng)計學(xué)意義(p0.05)。tce致敏組的b2rmrna表達水平也是逐漸增加的,且在72h時點達到最高。pksi致敏組的b1r以及b2rmrna表達水平都較tce致敏組的相應(yīng)時點相對降低。在72h時點組,tce致敏組比pksi致敏組的b1r和b2rmrna表達水平顯著增加,且差異有統(tǒng)計學(xué)意義(p0.05)。7.腎臟組織中b1r和b2r的蛋白表達水平采用westernblot法檢測腎臟組織中b1r以及b2r的蛋白表達水平。結(jié)果顯示:空白對照組與溶劑對照組的b1r蛋白幾乎不表達,b2r的表達也比較少。tce致敏組的b1r和b2r的表達明顯比溶劑對照組增加,且差異有統(tǒng)計學(xué)意義(p0.05)。兩個受體在tce致敏組中的表達也明顯比pksi致敏組多,差異有統(tǒng)計學(xué)意義(p0.05)。8.腎臟組織中c5b-9的沉積情況結(jié)果顯示如下:在空白對照組與溶劑對照組中c5b-9的表達都比較少,且差異無統(tǒng)計學(xué)意義(p0.05);在tce致敏組中24h、48h、72h沉積量逐漸增加,并在72h組達到最高峰,與溶劑對照組相比顯著升高,差異有統(tǒng)計學(xué)意義(p0.05),在7d時有所降低;pksi致敏組的四個時點的c5b-9表達含量都較TCE致敏組的相應(yīng)時點的表達明顯降低,且在48h和72h時點兩組差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1.一定劑量的TCE可引起小鼠皮膚致敏反應(yīng)并可導(dǎo)致小鼠的免疫性腎損傷。2.TCE致敏小鼠體內(nèi)補體激活的過程中還伴有KKS的活化,且KKS的活化及其產(chǎn)物在TCE所致的免疫性腎損傷過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.

【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】：2015
【分類號】：R135.7

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