亚砷酸钠诱导大鼠肝纤维化改变及相关差异蛋白表达的关联性研究

发布时间：2018-02-26 14:16

本文关键词： 砷代谢肝纤维化蛋白组学　出处：《新疆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:研究不同剂量亚砷酸钠慢性饮水染毒大鼠肝脏纤维化情况,寻找差异蛋白为进一步阐明砷致肝纤维化毒作用机制奠定基础。方法:将40只健康8周龄无特定病原体(Specefic pathogen Free,SPF)Sprague-Dawley(SD)雄性大鼠随机分为4组,分别为对照(去离子水)组及0.68、1.36、2.73mg/kg亚砷酸钠(iAs3+)染毒组。采用自由饮水方式进行染毒,连续染毒24周。收集24h尿样、血样,摘取肝脏,采用高效液相色谱-氢化物发生原子荧光光谱(HPLC-HGAFS)法测定大鼠肝脏及尿液总砷含量。采用酶联免疫分析法(ELISA)测定肝功能指标谷丙转氨酶(ALT)、谷草转氨酶(AST)和肝纤维化指标。制作肝组织切片,苏木精-伊红染色法(hematoxylin-eosin staining,HE)染色,光镜下观察病理变化情况,电子显微镜下观察细胞微结构变化。基于相对和绝对定量同位素标记(isobaric tags for relative and absolute quantitation,iTRAQ)的8标实验,结合2D LC-MS/MS对中、高剂量组与对照组大鼠肝脏组织蛋白表达差异进行比较。结果:1.亚砷酸钠染毒20周时高剂量组大鼠体重小于对照组(P0.05),24周时高剂量组大鼠体重小于对照组、低剂量组、中剂量组(P0.05),其余组间差异无统计学意义(P0.05)。2.中、高剂量组大鼠肝脏器系数与正常对照组相比升高,差异有统计学意义(P0.05),高剂量组与低剂量组肝脏器系数相比差异有统计学意义(P0.05),低剂量组与中剂量组差异无统计学意义(P0.05)。3.光、电镜下观察低剂量组未见明显的形态学改变,中、高剂量组随染毒剂量升高肝纤维化病变明显。4.UAs水平随染毒剂量增高而升高,其中,低、中、高剂量组与对照组相比,差异有统计学意义(P0.05),低剂量组UAs高于对照组,中剂量组高于低剂量组,高剂量组高于中剂量组,差异均有统计学意义(P0.05)。LAs组间差异有统计学意义(P0.05),随染毒剂量的增加,LAs水平升高,低剂量组LAs高于对照组,中剂量组高于低剂量组,高剂量组高于中剂量组,差异均有统计学意义(P0.05)。5.AST与ALT组间差异有统计学意义(P0.05),其中AST低剂量组与对照组差异无统计学意义;中、高剂量组均高于对照组和低剂量组(P0.05),中、高剂量组差异无统计学意义(P0.05)。ALT低、中、高剂量组高于对照组,低剂量组与中剂量组ALT含量差异无统计学意义(P0.05),其余组间比较均有统计学意义(P0.05),高剂量组高于低、中剂量组(P0.05)。6.“肝纤四项”指标组间差异有统计学意义(P0.05),两两比较发现HA、PCⅢ、LN三项指标均表现为低剂量组与对照组差异无统计学意义;中、高剂量组均高于对照组和低剂量组,差异有统计学意义,且高剂量组高于中剂量组(P0.05)。Ⅳ-C中、高剂量组高于对照组和低剂量组(P0.05),其余组间差异无统计学意义(P0.05)。7.iTRAQ技术结合2DLC-MS/MS技术选取蛋白质的置信阈值(Unused Prot Score)1.3,至少有1条匹配肽段在95%置信区间内作为蛋白质鉴定结果,鉴定到2948种蛋白质。文氏图比较发现三组均检测到的为2162种,除去对照组中也有意义的差异蛋白中剂量组上调蛋白为687种,下调为548种;高剂量组上调为633种,下调为519种。中、高剂量组表达差异蛋白数量,差异没有统计学意义(P0.05)。8.与甲基代谢相关的差异蛋白AS3MT、SHMT、BHMT、线粒体内CHDH、CTH以及CSAD,在中、高剂量组均上调;MTR有2种蛋白,其中METK1上调,F1LRB8下调,但高剂量组蛋白表达差异无意义。与GSH相关蛋白18种,包括Gsta1、Gsta4、Gsta5、Gstt1、Gstt2、Gstk1、Gstp1、Gstm1、Gstm2、Gstm3,Gss、Gpx1、Gpx4、Esd、Hagh、Glo1、Mgst1、B6DYQ5表达均上调;与肝纤维化有关的蛋白有Hic-5、Gss、Tpm,其中Tpm有6种包括Tpm1三种、Tpm2两种、Tpm3一种,表达均上调。结论:慢性砷暴露后砷在肝脏有蓄积,导致肝脏纤维化改变且肝功能下降,肝纤维化大鼠AS3MT、MTR、MAT、SHMT、BHMT、CHDH、CTH、CSAD、Hic-5、GSH、GSS、TPM高表达,可能在砷代谢和肝纤维化中发挥重要作用,与肝纤维化的发生相关。
[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.

【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114

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