本文選題：大鼠 + 急性肝損傷膽汁淤積模型�。� 參考：《昆明醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：通過α-萘異硫氰酸酯(ANIT)誘導(dǎo)SD大鼠急性肝損傷膽汁淤積模型,初步研究大鼠急性肝細(xì)胞損傷所致的膽汁淤積時肝臟組織中多藥耐藥相關(guān)蛋白2(MRP2)的表達(dá)變化。探討MRP2蛋白在急性肝損傷膽汁淤積模型中的表達(dá)與血清膽紅素及肝功能的相關(guān)性。 方法：100只健康成年雌性Sprague-Dawley(SD)大鼠作為實(shí)驗(yàn)對象,體重200±15g,隨機(jī)分為三組：即空白對照組(RK,n=30),條件對照組(RJ,n=30),中毒組(RP,n=40)。空白對照組不做任何處理；條件對照組按照5ml/kg予以灌服橄欖油；中毒組按1OOmg/kg(5ml/kg)一次性灌服ANIT-橄欖油溶液,所以大鼠恢復(fù)正常飲食。分別于灌服后24h、48h、72h、96h、120h五個時間點(diǎn)隨機(jī)挑選6只空白對照組,6只條件對照組及8只中毒組大鼠,麻醉后開腹,腹主動脈取血,檢測比較各組大鼠血清總膽紅素(TB)、丙氨酸氨基轉(zhuǎn)移酶(ALT)、天門冬氨酸氨基轉(zhuǎn)移酶(AST)水平。每只大鼠取2份肝組織樣本,一份提取肝組織總RNA,用逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)RT-PCR方法測定各組MRP2蛋白基因在肝臟組織中的表達(dá)水平；另一份采用HE染色觀察肝臟組織病理變化,采用SABC免疫組化方法測定在肝臟組織中MRP2蛋白的表達(dá)情況,并運(yùn)用Image Pro Plus6.0圖像分析軟件對累計光密度(IOD)值進(jìn)行半定量分析。 所得數(shù)據(jù)采用SPSS]7.0統(tǒng)計學(xué)軟件進(jìn)行數(shù)據(jù)分析,三組間比較采用單因素方差分析,p0.05認(rèn)為差異有統(tǒng)計學(xué)意義。本實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(x±s)描述。 結(jié)果：1.本實(shí)驗(yàn)成功利用ANIT建立了大鼠急性肝損傷膽汁淤積模型。 2.在三組大鼠血清膽紅素及肝功能的比較方面,RK組與RJ組之間差異無統(tǒng)計學(xué)意義(P0.05)；RP組與RK組及RJ組相比,各時間點(diǎn)血清TB、ALT、AST均顯著升高,并且均于48h達(dá)到高峰,之后逐漸下降,差異均有統(tǒng)計學(xué)意義(P0.05)。 3.RT-PCR結(jié)果中MRP2蛋白基因表達(dá)量的變化可見,RP組大鼠肝臟MRP2蛋白基因變化倍數(shù)(2(?)-ΔΔCt)明顯低于RK組和RJ組,差異有統(tǒng)計學(xué)意義(P0.05)。表明在大鼠肝損傷膽汁淤積模型中MRP2蛋白基因表達(dá)較正常明顯下降,其變化趨勢是：24h時即出現(xiàn)明顯下降,48h達(dá)到最低點(diǎn),之后緩慢上升,這與RP組大鼠血清膽紅素及酶學(xué)的變化呈現(xiàn)負(fù)相關(guān)性。 4.HE染色肝組織形態(tài)學(xué)觀察可見,RK組及RJ組肝小葉及匯管區(qū)結(jié)構(gòu)正常,無炎癥細(xì)胞浸潤,未見膽管增生。RP組可見,24h大鼠肝組織肝小葉及匯管區(qū)周邊出現(xiàn)炎性細(xì)胞浸潤,匯管區(qū)可見膽管上皮增厚；48h可見肝組織肝小葉及匯管區(qū)炎性細(xì)胞大量增多,膽管上皮較前明顯增厚,并可見少量的點(diǎn)、片狀壞死灶；之后肝損傷便逐漸修復(fù),至120h時肝組織炎癥反應(yīng)已基本消退,未見壞死灶。 5.免疫組化結(jié)果顯示：MRP2蛋白表達(dá)的陽性信號主要定位于細(xì)胞漿和細(xì)胞膜,以胞漿為主,呈棕黃色顆粒狀。RK組及RJ組可見MRP2蛋白染色均勻,表達(dá)正常,RP組可見染色變淺,且各時間點(diǎn)程度不同。利用IPP軟件測累計光密度值(IOD)行半定量分析,RP組IOD值較其余兩組明顯下降,且在48h達(dá)到最低值,差異有統(tǒng)計學(xué)意義(P0.05),表明RP組大鼠肝組織MRP2蛋白表達(dá)較正常明顯減少,且在48h達(dá)到最低值。這與RT-PCR的實(shí)驗(yàn)結(jié)果是基本一致的。 結(jié)論：ANIT所致的大鼠急性肝損傷膽汁淤積模型中,早期肝功能受損明顯,導(dǎo)致血清膽紅素及酶學(xué)水平明顯升高,MRP2蛋白的表達(dá)及其mRNA水平均逐步下降；48h之后隨著肝損傷的修復(fù),MRP2蛋白及其mRNA水平逐漸回升,膽紅素及酶學(xué)水平顯著下降,這說明MRP2的表達(dá)水平的變化與血清膽紅素及酶學(xué)的高低具有顯著的相關(guān)性,呈負(fù)相關(guān)。MRP2蛋白在對膽汁中膽紅素的排泄及肝臟的保護(hù)作用中具有重要意義。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.3

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