三氯乙烯致敏豚鼠肝组织中细胞因子TNF-α、IL-1、IL-6、IL-8水平研究

发布时间：2018-03-12 09:15

  本文选题：三氯乙烯　切入点：肝损伤　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的检测并比较不同组豚鼠肝组织中TNF-α、IL-1、IL-6、IL-8表达水平,研究细胞因子在TCE致敏豚鼠肝损伤中的作用,探讨三氯乙烯药疹样皮炎合并的肝损伤机制。 方法选用体重范围为250 g左右白色雌性豚鼠45只,随机分成空白对照组(5只)、溶剂对照组(5只)、TCE处理组(35只),依照豚鼠最大值实验(guinea pig maximization test,GPMT)方法建立动物致敏模型,并在终末激发后依据致敏结果以及取材时点不同(分别于终末激发后24h、72h取材),将TCE处理组分为TCE致敏24h组、72h组和TCE未致敏24h组、72h组。观察和记录受试区的皮肤反应情况并进行拍照,按表一对皮肤反应进行评分。抽取豚鼠血液并处死豚鼠,在无菌条件下迅速取下豚鼠肝脏,部分做成蜡块,部分做组织匀浆。于自动生化分析仪上检测肝功能,HE染色观察病理学改变,免疫组织化学方法以及酶联免疫吸附试验检测豚鼠皮肤肝组织中TNF-α、IL-1、IL-6和IL-8的表达水平,用SPSS 13.0统计软件包对实验结果进行统计分析。 结果 1.在此次皮肤致敏反应试验中,空白组和溶剂对照组均未见红斑或水肿;TCE处理组的30只豚鼠中有19只出现红斑,致敏率为63.3%。 2.肝功能TCE致敏组的ALT、AST水平较溶剂对照组升高,有统计学意义(p0.05);TCE致敏72h组ALT、AST水平显著高于TCE致敏24h组以及TCE14d组,且有统计学意义(p0.05);TCE致敏72h组ALB水平下降,与溶剂对照组比较有统计学意义(p0.05)。 3.病理学观察空白对照组、溶剂对照组豚鼠肝细胞排列整齐,胞质均匀,胞核大而圆,核仁明显,核膜清晰; TCE未致敏组能偶见肝细胞水肿;TCE致敏24h组中能够见到少量肝细胞水肿;TCE致敏72h组中能够见到较多肝细胞水肿,呈空泡样改变,并有胞核破裂消失。 4.肝组织中TNF-α、IL-1、IL-6、IL-8水平检测 4.1 TNF-α检测结果免疫组化结果:TCE14d组、TCE致敏24h组、TCE致敏72h组TNF-α水平较溶剂对照组上升(p0.05);TCE致敏组肝组织中TNF-α水平分别高于各自未致敏组(p0.05);TCE致敏72h组肝组织中TNF-α水平要高于TCE致敏24h组以及TCE14d组(p0.05)。ELISA结果:TCE未致敏24h组、TCE致敏组24h组、TCE未致敏72h组、TCE致敏72h组肝组织中TNF-α水平高于溶剂对照组(p0.05);TCE致敏24h组、TCE致敏72h组肝组织中TNF-α水平分别高于各自未致敏组(p0.05);TCE致敏72h组、TCE致敏24h组肝组织中TNF-α水平均高于TCE14d组水平p0.05)。 4.2 IL-1检测结果免疫组化结果:与溶剂对照组比较,TCE致敏24h组、TCE致敏72h组以及TCE未致敏24h组肝组织中IL-1β水平升高(p0.05);TCE致敏组24h、TCE致敏72h组肝组织中IL-1β水平分别高于各自未至敏组(p0.05);TCEh致敏24组、TCE致敏72h组肝组织中IL-1β水平均高于T14d组(p0.05);而且,TCE致敏72h组肝组织中IL-1β水平要高于TCE致敏24h组(p0.05)。ELISA结果:TCE处理的各组肝组织中IL-1水平较溶剂对照组都上升,除TCE未致敏72h组外,其它组有统计学意义(p0.05);TCE致敏72h组肝组织中IL-1水平高于TCE未致敏72h组(p0.05);TCE致敏72h组肝组织中IL-1水平高于TCE致敏24h组、TCE14d组(p0.05)。 4.3 IL-6检测结果免疫组化结果:TCE致敏24h组、TCE致敏72h组肝组织中IL-6水平较溶剂对照组升高(p0.05);TCE致敏24h组、TCE致敏72h组肝组织中IL-6水平分别高于各自未至敏组(p0.05);TCE致敏24h组、TCE致敏72h组肝组织中IL-6水平均高于T14d组(p0.05)。ELISA结果:TCE致敏组24h组、TCE未致敏72h组、TCE致敏72h组较溶剂对照组升高,差异且有统计学意义(p0.05);TCE致敏72h组肝组织中IL-6水平高于TCE未致敏72h组,差异有统计学意义(p0.05);TCE致敏72h组、TCE致敏24h组肝组织中IL-6水平均高于TCE14d组水平,其中TCE致敏72h组有统计学意义(p0.05)。 4.4 IL-8检测结果免疫组化结果:与溶剂对照比较,TCE致敏24h组、TCE致敏72h组肝组织中IL-8水平升高(p0.05);TCE致敏24h组、TCE致敏72h组肝组织中IL-8水平分别高于各自未致敏组(p0.05);TCE致敏24h组、TCE致敏72h组肝组织中IL-8水平均高于T14d组(p0.05)。ELISA结果:TCE处理的各组肝组织中IL-8水平都上升,且有统计学意义(p0.05);TCE致敏72h组肝组织中IL-8水平高于TCE未致敏72h组,差异有统计学意义(p0.05);TCE致敏72h组、TCE致敏24h组肝组织中IL-8水平均高于TCE14d组水平,其中TCE致敏72h组有统计学意义(p0.05)。 5.肝组织中细胞因子以及细胞因子与肝功能间相关性分析 TNF-α与IL-1、IL-6、IL-8之间存在显著正相关,即随着肝组织中TNF-α水平的上升,肝组织中IL-1、IL-6、IL-8的表达也增加;IL-1与IL-6、IL-8之间也存在显著正相关,但IL-6与IL-8相关性无统计学意义。ALT、AST与TNF-α、IL-6呈正相关,且有统计学意义(p0.05);除与TNF-α呈负相关有统计学意义(p0.05),ALB与IL-1、IL-6、IL-8相关性无统计学意义。 结论:本研究证实TCE对豚鼠具有致敏作用;能够引起致敏豚鼠不同程度的肝损伤;TCE致敏豚鼠肝组织内TNF-α、IL-1、IL-6、IL-8水平较其它组高,且ALT、AST与TNF-α、IL-6呈正相关,提示TNF-α、IL-1、IL-6、IL-8在三氯乙烯致敏豚鼠肝损伤的过程中发挥重要作用。
[Abstract]:Objective to detect and compare the expression levels of TNF-, IL-1, IL-6 and IL-8 in different groups of guinea pig liver tissues, and investigate the role of cytokines in the liver injury induced by TCE sensitized guinea pigs, and to explore the mechanism of liver injury associated with trichloroethylene induced drug eruption dermatitis.
Methods the weight range is about 250 g 45 white female guinea pigs were randomly divided into control group (5 rats), solvent control group (5 rats), TCE group (35), in accordance with the maximum value of experimental guinea pigs (guinea pig maximization test, GPMT) method to establish animal model of sensitization, and in the end at the end of excitation according to sensitization results and different sampling point (respectively excited at the terminal of 24h, 72h, were) TCE treatment group was divided into TCE sensitized 24h group, 72h group and TCE sensitized 24h group and 72h group. Observe and record the skin reaction test area and take photos, according to a table of skin reaction score. Extraction of guinea pig blood and guinea pigs were sacrificed, quickly remove the guinea pig liver under aseptic conditions, some wax blocks, part of homogenate. In automatic biochemical analyzer to detect liver function, pathological changes were observed with HE staining, immunohistochemistry and enzyme-linked immunosorbent assay The expression level of TNF- alpha, IL-1, IL-6 and IL-8 in the skin liver tissues of guinea pigs was detected, and the experimental results were statistically analyzed with the statistical package of SPSS 13.
Result
1. in the skin sensitization test, there were no erythema or edema in the blank group and the solvent control group. 19 of the 30 guinea pigs in the TCE treated group had erythema, and the sensitization rate was 63.3%..
2. liver function TCE ALT sensitized group, AST level compared with the solvent control group increased, with statistical significance (P0.05); TCE sensitized 72h group ALT, AST was significantly higher than that of TCE sensitized 24h group and TCE14d group, and there was statistical significance (P0.05); TCE sensitized 72h group ALB decreased, and the solvent the control group was statistically significant (P0.05).
Observation of the blank control group, solvent control group 3. pathology of guinea pig liver cells arranged in neat, uniform cytoplasm, large round nucleus, obvious nucleolus, nuclear membrane was clear; TCE sensitized group can even see liver cell edema; TCE sensitized 24h group to be able to see a small amount of liver cell edema; TCE sensitized 72h group to see more liver cell edema, vacuolar changes, and nucleus broken away.
Detection of TNF- alpha, IL-1, IL-6, IL-8 in 4. liver tissues
4.1 TNF- alpha test results the results of immunohistochemistry: TCE14d group, TCE sensitized 24h group and TCE sensitized 72h group TNF- levels than the solvent control group increased (P0.05); TCE TNF- induced by alpha level of liver tissue were higher than their respective sensitive sensitized group (P0.05); TCE TNF- induced by alpha level of liver sensitive tissue in the 72h group than the TCE sensitized 24h group and TCE14d group (P0.05.ELISA) results: TCE sensitized 24h group and TCE sensitized group 24h group TCE sensitized 72h group, TCE induced TNF- levels of liver tissue sensitivity to 72h was significantly higher than that of the solvent control group (P0.05); TCE sensitization 24h group, TCE induced TNF- levels of liver tissue in 72h group were higher than that of their sensitive non sensitized group (P0.05); TCE sensitized 72h group, TCE induced TNF- levels of liver tissue sensitivity was higher in group 24h than in group TCE14d P0.05).
4.2 IL-1妫，

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