MRP2在大鼠急性肝损伤胆汁淤积模型中的表达与血清胆红素及肝功能的相关性研究

发布时间：2018-04-26 10:45

本文选题：大鼠 + 急性肝损伤胆汁淤积模型　；参考：《昆明医科大学》2014年硕士论文

【摘要】：目的：通过α-萘异硫氰酸酯(ANIT)诱导SD大鼠急性肝损伤胆汁淤积模型,初步研究大鼠急性肝细胞损伤所致的胆汁淤积时肝脏组织中多药耐药相关蛋白2(MRP2)的表达变化。探讨MRP2蛋白在急性肝损伤胆汁淤积模型中的表达与血清胆红素及肝功能的相关性。方法：100只健康成年雌性Sprague-Dawley(SD)大鼠作为实验对象,体重200±15g,随机分为三组：即空白对照组(RK,n=30),条件对照组(RJ,n=30),中毒组(RP,n=40)。空白对照组不做任何处理；条件对照组按照5ml/kg予以灌服橄榄油；中毒组按1OOmg/kg(5ml/kg)一次性灌服ANIT-橄榄油溶液,所以大鼠恢复正常饮食。分别于灌服后24h、48h、72h、96h、120h五个时间点随机挑选6只空白对照组,6只条件对照组及8只中毒组大鼠,麻醉后开腹,腹主动脉取血,检测比较各组大鼠血清总胆红素(TB)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平。每只大鼠取2份肝组织样本,一份提取肝组织总RNA,用逆转录-聚合酶链式反应RT-PCR方法测定各组MRP2蛋白基因在肝脏组织中的表达水平；另一份采用HE染色观察肝脏组织病理变化,采用SABC免疫组化方法测定在肝脏组织中MRP2蛋白的表达情况,并运用Image Pro Plus6.0图像分析软件对累计光密度(IOD)值进行半定量分析。所得数据采用SPSS]7.0统计学软件进行数据分析,三组间比较采用单因素方差分析,p0.05认为差异有统计学意义。本实验数据用均数±标准差(x±s)描述。结果：1.本实验成功利用ANIT建立了大鼠急性肝损伤胆汁淤积模型。 2.在三组大鼠血清胆红素及肝功能的比较方面,RK组与RJ组之间差异无统计学意义(P0.05)；RP组与RK组及RJ组相比,各时间点血清TB、ALT、AST均显著升高,并且均于48h达到高峰,之后逐渐下降,差异均有统计学意义(P0.05)。 3.RT-PCR结果中MRP2蛋白基因表达量的变化可见,RP组大鼠肝脏MRP2蛋白基因变化倍数(2(?)-ΔΔCt)明显低于RK组和RJ组,差异有统计学意义(P0.05)。表明在大鼠肝损伤胆汁淤积模型中MRP2蛋白基因表达较正常明显下降,其变化趋势是：24h时即出现明显下降,48h达到最低点,之后缓慢上升,这与RP组大鼠血清胆红素及酶学的变化呈现负相关性。 4.HE染色肝组织形态学观察可见,RK组及RJ组肝小叶及汇管区结构正常,无炎症细胞浸润,未见胆管增生。RP组可见,24h大鼠肝组织肝小叶及汇管区周边出现炎性细胞浸润,汇管区可见胆管上皮增厚；48h可见肝组织肝小叶及汇管区炎性细胞大量增多,胆管上皮较前明显增厚,并可见少量的点、片状坏死灶；之后肝损伤便逐渐修复,至120h时肝组织炎症反应已基本消退,未见坏死灶。 5.免疫组化结果显示：MRP2蛋白表达的阳性信号主要定位于细胞浆和细胞膜,以胞浆为主,呈棕黄色颗粒状。RK组及RJ组可见MRP2蛋白染色均匀,表达正常,RP组可见染色变浅,且各时间点程度不同。利用IPP软件测累计光密度值(IOD)行半定量分析,RP组IOD值较其余两组明显下降,且在48h达到最低值,差异有统计学意义(P0.05),表明RP组大鼠肝组织MRP2蛋白表达较正常明显减少,且在48h达到最低值。这与RT-PCR的实验结果是基本一致的。结论：ANIT所致的大鼠急性肝损伤胆汁淤积模型中,早期肝功能受损明显,导致血清胆红素及酶学水平明显升高,MRP2蛋白的表达及其mRNA水平均逐步下降；48h之后随着肝损伤的修复,MRP2蛋白及其mRNA水平逐渐回升,胆红素及酶学水平显著下降,这说明MRP2的表达水平的变化与血清胆红素及酶学的高低具有显著的相关性,呈负相关。MRP2蛋白在对胆汁中胆红素的排泄及肝脏的保护作用中具有重要意义。
[Abstract]:Objective: To investigate the expression of multidrug resistance associated protein 2 (MRP2) in the liver tissues of rats with acute hepatic injury induced by acute hepatocyte injury by alpha naphthyl cyanate (ANIT) induced cholestasis of acute hepatic injury in SD rats. The expression of MRP2 protein in the cholestasis model of acute liver injury and the serum bilirubin were discussed. And the correlation of liver function.
Methods: 100 healthy adult female Sprague-Dawley (SD) rats were randomly divided into three groups: the blank control group (RK, n=30), the condition control group (RJ, n=30), the toxic group (RP, n=40). The control group was given the olive oil in the control group and the control group was given 1OOmg/kg (5ml/kg) in 1OOmg/kg (5ml/kg). The rats were given ANIT- olive oil solution at one time, so the rats returned to normal diet. 6 blank control groups were randomly selected at five time points of 24h, 48h, 72h, 96h and 120h, respectively, 6 condition control groups and 8 rats, the abdominal aorta was taken after anesthesia, and the serum total bilirubin (TB) and alanine aminotransferase was detected. Enzyme (ALT), aspartate aminotransferase (AST) level. 2 liver tissue samples were taken in each rat, one liver tissue total RNA was extracted, and the expression level of MRP2 protein gene in liver tissues was measured by reverse transcription polymerase chain reaction (RT-PCR); the other used HE staining to observe the pathological changes of liver tissue, and the SABC immunization group was used. The expression of MRP2 protein in liver tissues was determined by chemical method, and the value of cumulative optical density (IOD) was semi quantitative analyzed by Image Pro Plus6.0 image analysis software.
The data were analyzed by SPSS]7.0 statistics software, and the three groups were compared with single factor analysis of variance. P0.05 thought the difference was statistically significant. The experimental data were described with mean standard deviation (x + s).
Results: 1. the cholestasis model of rats with acute liver injury was successfully established by ANIT.
2. in the comparison of serum bilirubin and liver function in the three groups, there was no significant difference between the RK group and the RJ group (P0.05). The serum TB, ALT and AST in the RP group were significantly higher than those in the RK group and the RJ group at all time points, and all reached the peak in 48h, and then decreased gradually, and the difference was statistically significant (P0.05).
The changes in the expression of MRP2 protein gene in 3.RT-PCR showed that the MRP2 protein gene change multiple (2 (?) - Delta Ct) in the rat liver was significantly lower than that of the RK group and the RJ group, and the difference was statistically significant (P0.05). It showed that the expression of the MRP2 protein gene in the rat liver injury cholestasis model was significantly lower than that in the normal rat liver injury model. The trend of the change is: 24h is the emergence of 24h. Significantly decreased, 48h reached the lowest point and then slowly increased, which was negatively correlated with the changes of serum bilirubin and enzymology in RP rats.
4.HE staining liver histomorphology observation showed that the structure of liver lobules and sinks in group RK and RJ was normal, no inflammatory cell infiltration, no bile duct hyperplasia.RP group, inflammatory cell infiltration in hepatic lobule and peripheral area of liver tissue of 24h rats, thickening of bile duct epithelium, and 48h visible hepatic lobules and inflammatory cells in confluence area. In a large amount, the bile duct epithelium is thicker than before, and a small number of points and flaky necrotic foci are seen, and the liver injury is gradually repaired. The inflammatory reaction of the liver tissue has basically subsided and no necrotic focus is found at the time of 120h.
5. the results of immunohistochemical staining showed that the positive signal of MRP2 protein expression was mainly located in the cytoplasm and cell membrane, mainly in the cytoplasm, in the brown yellow granular.RK group and in the RJ group, the MRP2 protein staining was uniform and the expression was normal. The RP group showed the light staining and the different time points. The accumulated light density value (IOD) was half quantified by the IPP software. The IOD value of group RP was significantly lower than that of the other two groups, and the lowest value in 48h was reached, and the difference was statistically significant (P0.05). It showed that the expression of MRP2 protein in the liver tissue of the RP group was significantly reduced and the lowest value in 48h. This was basically consistent with the experimental results of RT-PCR.
Conclusion: in the model of acute liver injury induced by ANIT in rats, the early liver function was damaged obviously, the level of serum bilirubin and enzymology increased obviously, the expression of MRP2 protein and the level of mRNA decreased gradually. After the repair of liver injury, the level of MRP2 protein and its mRNA increased gradually, and the level of bilirubin and enzyme was significant. This indicates that the changes in the expression level of MRP2 have a significant correlation with the level of serum bilirubin and enzyme, and the negative correlation of.MRP2 protein is of great significance in the excretion of bilirubin in bile and the protection of the liver.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.3

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