本文關鍵詞： 砷代謝 肝纖維化 蛋白組學　出處：《新疆醫(yī)科大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的:研究不同劑量亞砷酸鈉慢性飲水染毒大鼠肝臟纖維化情況,尋找差異蛋白為進一步闡明砷致肝纖維化毒作用機制奠定基礎。方法:將40只健康8周齡無特定病原體(Specefic pathogen Free,SPF)Sprague-Dawley(SD)雄性大鼠隨機分為4組,分別為對照(去離子水)組及0.68、1.36、2.73mg/kg亞砷酸鈉(iAs3+)染毒組。采用自由飲水方式進行染毒,連續(xù)染毒24周。收集24h尿樣、血樣,摘取肝臟,采用高效液相色譜-氫化物發(fā)生原子熒光光譜(HPLC-HGAFS)法測定大鼠肝臟及尿液總砷含量。采用酶聯(lián)免疫分析法(ELISA)測定肝功能指標谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)和肝纖維化指標。制作肝組織切片,蘇木精-伊紅染色法(hematoxylin-eosin staining,HE)染色,光鏡下觀察病理變化情況,電子顯微鏡下觀察細胞微結(jié)構(gòu)變化�；谙鄬徒^對定量同位素標記(isobaric tags for relative and absolute quantitation,iTRAQ)的8標實驗,結(jié)合2D LC-MS/MS對中、高劑量組與對照組大鼠肝臟組織蛋白表達差異進行比較。結(jié)果:1.亞砷酸鈉染毒20周時高劑量組大鼠體重小于對照組(P0.05),24周時高劑量組大鼠體重小于對照組、低劑量組、中劑量組(P0.05),其余組間差異無統(tǒng)計學意義(P0.05)。2.中、高劑量組大鼠肝臟器系數(shù)與正常對照組相比升高,差異有統(tǒng)計學意義(P0.05),高劑量組與低劑量組肝臟器系數(shù)相比差異有統(tǒng)計學意義(P0.05),低劑量組與中劑量組差異無統(tǒng)計學意義(P0.05)。3.光、電鏡下觀察低劑量組未見明顯的形態(tài)學改變,中、高劑量組隨染毒劑量升高肝纖維化病變明顯。4.UAs水平隨染毒劑量增高而升高,其中,低、中、高劑量組與對照組相比,差異有統(tǒng)計學意義(P0.05),低劑量組UAs高于對照組,中劑量組高于低劑量組,高劑量組高于中劑量組,差異均有統(tǒng)計學意義(P0.05)。LAs組間差異有統(tǒng)計學意義(P0.05),隨染毒劑量的增加,LAs水平升高,低劑量組LAs高于對照組,中劑量組高于低劑量組,高劑量組高于中劑量組,差異均有統(tǒng)計學意義(P0.05)。5.AST與ALT組間差異有統(tǒng)計學意義(P0.05),其中AST低劑量組與對照組差異無統(tǒng)計學意義;中、高劑量組均高于對照組和低劑量組(P0.05),中、高劑量組差異無統(tǒng)計學意義(P0.05)。ALT低、中、高劑量組高于對照組,低劑量組與中劑量組ALT含量差異無統(tǒng)計學意義(P0.05),其余組間比較均有統(tǒng)計學意義(P0.05),高劑量組高于低、中劑量組(P0.05)。6.“肝纖四項”指標組間差異有統(tǒng)計學意義(P0.05),兩兩比較發(fā)現(xiàn)HA、PCⅢ、LN三項指標均表現(xiàn)為低劑量組與對照組差異無統(tǒng)計學意義;中、高劑量組均高于對照組和低劑量組,差異有統(tǒng)計學意義,且高劑量組高于中劑量組(P0.05)。Ⅳ-C中、高劑量組高于對照組和低劑量組(P0.05),其余組間差異無統(tǒng)計學意義(P0.05)。7.iTRAQ技術結(jié)合2DLC-MS/MS技術選取蛋白質(zhì)的置信閾值(Unused Prot Score)1.3,至少有1條匹配肽段在95%置信區(qū)間內(nèi)作為蛋白質(zhì)鑒定結(jié)果,鑒定到2948種蛋白質(zhì)。文氏圖比較發(fā)現(xiàn)三組均檢測到的為2162種,除去對照組中也有意義的差異蛋白中劑量組上調(diào)蛋白為687種,下調(diào)為548種;高劑量組上調(diào)為633種,下調(diào)為519種。中、高劑量組表達差異蛋白數(shù)量,差異沒有統(tǒng)計學意義(P0.05)。8.與甲基代謝相關的差異蛋白AS3MT、SHMT、BHMT、線粒體內(nèi)CHDH、CTH以及CSAD,在中、高劑量組均上調(diào);MTR有2種蛋白,其中METK1上調(diào),F1LRB8下調(diào),但高劑量組蛋白表達差異無意義。與GSH相關蛋白18種,包括Gsta1、Gsta4、Gsta5、Gstt1、Gstt2、Gstk1、Gstp1、Gstm1、Gstm2、Gstm3,Gss、Gpx1、Gpx4、Esd、Hagh、Glo1、Mgst1、B6DYQ5表達均上調(diào);與肝纖維化有關的蛋白有Hic-5、Gss、Tpm,其中Tpm有6種包括Tpm1三種、Tpm2兩種、Tpm3一種,表達均上調(diào)。結(jié)論:慢性砷暴露后砷在肝臟有蓄積,導致肝臟纖維化改變且肝功能下降,肝纖維化大鼠AS3MT、MTR、MAT、SHMT、BHMT、CHDH、CTH、CSAD、Hic-5、GSH、GSS、TPM高表達,可能在砷代謝和肝纖維化中發(fā)揮重要作用,與肝纖維化的發(fā)生相關。
[Abstract]:Objective: To study the liver fibrosis of different doses of sodium arsenite in rats exposed to chronic drinking water situation, find the differences of the protein to lay the foundation for the toxic mechanism of hepatic fibrosis to further elucidate arsenic. Methods: 40 healthy 8 week old specific pathogen free (Specefic pathogen, Free, SPF) Sprague-Dawley (SD) male rats were randomly divided into 4 groups respectively, the control group 0.68,1.36,2.73mg/kg (deionized water) and sodium arsenite (iAs3+) exposure group. Through drinking water exposure, after 24 weeks. Collect 24h urine, blood, liver, using high performance liquid chromatography hydride generation atomic fluorescence spectrometry (HPLC-HGAFS) method for the determination of total arsenic in urine and liver of rats content. By enzyme-linked immunosorbent assay (ELISA) determination of liver function indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic fibrosis. Making the liver tissue slices, hematoxylin eosin staining (hematoxyli N-eosin staining, HE) staining, light microscope to observe the pathological changes and cell microstructure changes observed under electron microscope. The relative and absolute quantification based on isotope labeling (isobaric tags for relative and absolute quantitation, iTRAQ) of the 8 calibration experiments, combined with the 2D LC-MS/ of MS, compared with the high dose group and the liver tissue protein group differential expression of control rats. Results: 1. sodium arsenite for 20 weeks when the weight of rats in high dose group than the control group (P0.05), 24 weeks in high dose group rats weight less than the control group, low dose group, middle dose group (P0.05), no statistically significant differences between the other groups (P0.05.2.) in the coefficient of liver of rats in high dose group compared with normal control group increased, the difference was statistically significant (P0.05), high dose group compared with the low dose group liver coefficient was statistically significant difference (P0.05), low dose group and middle dose group were no significant Statistically significant (P0.05).3. light, electron microscope observation, there was no obvious morphological change, low dose group, high dose group increased withexposure dose increased significantly.4.UAs level of liver fibrosis with the dose increased, the low and high dose group compared with the control group, the difference was statistically significant (P0.05), low dose of UAs group was higher than the control group, middle dose group was higher than that of low dose group, high dose group was higher than that of middle dose group, the differences were statistically significant (P0.05) difference was statistically significant between group.LAs (P0.05), with the dose increased, the elevated levels of LAs and LAs in low dose group than the control group, middle dose group was higher than that of low dose group, high dose group was higher than that of middle dose group, the differences were statistically significant (P0.05) difference was statistically significant between group.5.AST and ALT (P0.05), the AST low dose group and the control group no significant difference; in high dose group were higher than those in control group and The low dose group (P0.05), and no significant difference between the high dose group (P0.05), low.ALT, high dose group was higher than the control group, no statistically significant difference between the low dose group and middle dose group ALT content (P0.05), the other groups were statistically significant (P0.05), high dose group was higher than that of low the middle dose group (P0.05), there was statistical significance in.6. "Ganxian four" indicators of differences between the groups (P0.05), 22 HA, PC III, LN three indicators showed that low dose group and the control group had no significant difference; in high dose group were higher than those of control group and the low dose group, the difference was statistically significant, and the high dose group was higher than that of middle dose group (P0.05). 4 -C, the high dose group was higher than control group and low dose group (P0.05), no statistically significant differences between the other groups (P0.05) confidence threshold of.7.iTRAQ technology combined with 2DLC-MS/MS technology to select protein (Unused Prot Score) 1.3, at least 1 A matching peptide in 95% confidence interval as a result of protein identification, identification of 2948 proteins. Comparing the Venn diagram three groups were detected for 2162, remove the dose group in the control group have significance difference in protein up-regulated protein 687, down to 548; the high dose group increased to 633 a cut to 519. In the high dose group differences in protein expression quantity, the difference was not statistically significant difference (P0.05) of.8. and methyl metabolism proteins AS3MT, SHMT, BHMT, CHDH, CTH and CSAD in the mitochondria, and in high dose group were up-regulated; MTR 2 proteins, including METK1 up-regulated however, the down-regulation of F1LRB8 protein expression in high dose group, there was no significant difference between GSH. 18 genes, including Gsta1, Gsta4, Gsta5, Gstt1, Gstt2, Gstk1, Gstp1, Gstm1, Gstm2, Gstm3, Gss, Gpx1, Gpx4, Esd, Hagh, Glo1, Mgst1, B6DYQ5 were up-regulated in the liver; and the protein of Hic-5 G fibrosis. SS, Tpm, Tpm which has 6 kinds including Tpm1 three, Tpm2 two, Tpm3 a, were up-regulated. Conclusion: chronic exposure to arsenic in the liver after arsenic accumulation, resulting in liver fibrosis and liver function decline, AS3MT, rat liver fibrosis MTR, MAT, SHMT, BHMT, CHDH, CTH CSAD, Hic-5, GSH, GSS, TPM, high expression may play an important role in arsenic metabolism and liver fibrosis, and liver fibrosis.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R114

【參考文獻】

相關期刊論文 前1條

1 李秋娟;楊光;葉建新;孫鮮策;陳敏;劉曉芳;樸豐源;;砷暴露小鼠肝組織核酸損傷免疫組織化學觀察[J];中國公共衛(wèi)生;2007年04期

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本文編號：1538357