本文選題：DEN　切入點(diǎn)：肝纖維化　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：肝癌(liver cancer)是最常見(jiàn)威脅人類健康的肝臟惡性腫瘤。肝炎-肝硬化-肝癌,這一發(fā)展模式得到支持。肝纖維化是各種慢性肝病共同的病理學(xué)特征,也是慢性肝病進(jìn)展到肝硬化的必經(jīng)階段。乙醇脫氫酶(alcohol dehydrogenases,ADH)和乙醛脫氫酶(aldehyde dehydrogenases,ALDH)主要存在于肝細(xì)胞中。ADH主要參與乙醇代謝,乙醇經(jīng)氧化生成乙醛,乙醛具有肝臟毒性,能誘發(fā)癌癥。同時(shí)ADH也參與視黃醇代謝,視黃醇及其代謝物參與肝炎及肝纖維化的進(jìn)程,近期結(jié)果也顯示ADH的缺乏能抑制肝纖維化。據(jù)報(bào)道,和癌旁的正常組織相比,肝癌組織中ADHI活性出現(xiàn)升高。肝癌患者血清中ADH活性較健康人也出現(xiàn)升高。本室前期結(jié)果顯示,和正常肝組織相比,肝癌患者的肝纖維化組織中總ADH、ADHI、ADHII酶活性均明顯升高,而總ALDH和ALDH2酶活性沒(méi)有改變。上述結(jié)果顯示,ADH活性升高可能和肝纖維化甚至肝癌存在關(guān)系,但肝癌發(fā)展的各個(gè)階段血清和組織中ADH活性是如何改變的,ADH活性的升高是肝損傷的結(jié)果還是原因,其升高是否存在對(duì)肝損傷的易感性有待實(shí)驗(yàn)進(jìn)一步研究。本實(shí)驗(yàn)擬選擇二乙基亞硝胺(diethylnitrosamine,DEN)制備的大鼠肝癌模型為研究對(duì)象,測(cè)定大鼠在誘導(dǎo)的不同階段ADH活性,分析大鼠血漿中ADH基礎(chǔ)活性和肝損傷相關(guān)指標(biāo)的相關(guān)性,并分析肝組織中ADH、ALDH活性和肝損傷相關(guān)指標(biāo)的相關(guān)性,以期探討ADH在肝纖維化甚至肝癌發(fā)展中所起的作用。方法1大鼠肝癌模型建立1.1動(dòng)物分組和給藥方法54只SD雄性大鼠,分成2組,對(duì)照組10只,模型組44只,適應(yīng)性喂養(yǎng)后,模型組1-4周腹腔注射50 mg/kg DEN,每周兩次,5-14周改為腹腔注射50mg/kg DEN,每周一次,在誘導(dǎo)至12周時(shí)隨機(jī)處死20只模型組大鼠,命名為模型A組。剩余大鼠繼續(xù)按照上述方法給藥,14周給藥結(jié)束后繼續(xù)飼養(yǎng),于19周處死,此組大鼠命名為模型B組。對(duì)照組10只大鼠在19周處死。1.2動(dòng)物觀察與取材每天觀察大鼠生理和精神狀態(tài),每周記錄大鼠體重,對(duì)照組和模型B組于給藥前第0周,及開(kāi)始給藥后第8、12、16、19周留取大鼠血漿。模型A組于給藥前第0周,及開(kāi)始給藥后第8、12周留取大鼠血漿。大鼠處死后,取出肝臟,肝組織一部分進(jìn)行病理檢查,剩余部分放入液氮。1.3肝勻漿的制備及蛋白濃度測(cè)定稱取適量肝組織,加入9倍體積的勻漿介質(zhì),制成10%組織勻漿,離心取上清,用BCA法測(cè)定肝勻漿蛋白濃度。1.4血漿和肝勻漿中ALT、AST活性測(cè)定血漿和肝勻漿中谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)活性參照試劑盒說(shuō)明書通過(guò)酶標(biāo)儀測(cè)定。1.5肝組織病理染色測(cè)定對(duì)照組和A、B模型組大鼠肝組織切片進(jìn)行HE、masson染色,并根據(jù)Ishak評(píng)分系統(tǒng)對(duì)肝標(biāo)本進(jìn)行分級(jí),并測(cè)定了masson染色后纖維化面積占比。1.6肝組織免疫組化測(cè)定通過(guò)免疫組化方法測(cè)定對(duì)照組和A、B模型組大鼠細(xì)胞增殖指數(shù)(Ki67)、增殖細(xì)胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)、胎盤型谷胱甘肽硫轉(zhuǎn)移酶(Glutathione S-transferase placental form,GST-p)蛋白的表達(dá)水平和表達(dá)位置。2血漿ADH和肝勻漿ADH、ALDH活性測(cè)定ADH催化氧化型輔酶Ⅰ(NAD+)反應(yīng)生成還原型輔酶I(NADH),NADH在340 nm處有吸收,通過(guò)測(cè)定340 nm處吸光度變化得到ADH活性,血漿和肝勻漿中ADH活性用此原理的試劑盒測(cè)定。ALDH催化NAD+反應(yīng)生成NADH,測(cè)定340 nm處吸光度變化得到ALDH活性。肝勻漿中ALDH活性用此原理試劑盒測(cè)定。3統(tǒng)計(jì)方法采用SPSS17.0軟件對(duì)各項(xiàng)數(shù)據(jù)資料進(jìn)行統(tǒng)計(jì)學(xué)分析。兩組間數(shù)據(jù)的比較采用獨(dú)立樣本t檢驗(yàn),三組及三組以上數(shù)據(jù)的比較用單因素方差分析,ADH、ALDH活性、ADH/ALDH與肝損傷相關(guān)指標(biāo)間的相關(guān)性采用Person相關(guān),檢驗(yàn)水準(zhǔn)α為0.05。結(jié)果1大鼠肝癌模型建立1.1大鼠體重肝重與對(duì)照組相比,模型組大鼠體重從誘模第2周到處死時(shí)均下降(P0.001)。和對(duì)照組相比,模型A組肝重?zé)o明顯改變(P0.05),肝重/體重明顯升高(P0.001);模型B組大鼠肝重升高(P0.001),肝重/體重升高(P0.001)。1.2大鼠存活情況模型組大鼠在誘導(dǎo)的前12周無(wú)死亡,在15-19周陸續(xù)死亡14只。1.3血漿和肝勻漿中ALT、AST活性與對(duì)照組相比,在8、12、16和19周模型組大鼠血漿中ALT、AST活性均有明顯升高(P0.001)。與對(duì)照組相比,模型A組大鼠肝勻漿中ALT活性下降(P0.05),模型B組大鼠肝勻漿中ALT活性下降更明顯(P0.01);與對(duì)照組相比,模型A組大鼠肝勻漿中AST活性升高(P0.05),模型B組大鼠肝勻漿中AST活性升高更明顯(P0.01);與對(duì)照組相比,A、B模型組AST/ALT比值均升高(P0.001)。1.4病理染色結(jié)果和成癌率與對(duì)照組相比,A、B模型組大鼠肝組織Ishak評(píng)分和masson染色后纖維化所占面積均明顯升高(P0.001)。模型A組(12周處死)大鼠均出現(xiàn)不同程度的肝纖維化。模型B組(19周處死)有5只大鼠出現(xiàn)肝腺瘤,9只出現(xiàn)肝細(xì)胞癌,成癌率為66.7%。1.5肝損傷相關(guān)指標(biāo)的結(jié)果與對(duì)照組相比,A、B模型組大鼠肝臟切片中Ki67、PCNA陽(yáng)性細(xì)胞率和GST-p平均光密度值均增加(P0.01)。與模型A組相比,模型B組Ki67和PCNA陽(yáng)性細(xì)胞率下降(P0.01),GST-p平均光密度值無(wú)明顯改變(P0.05)。2大鼠ADH、ALDH活性2.1大鼠血漿中ADH活性與對(duì)照組相比,模型組大鼠血漿中ADH活性在16周(24.21±9.86 U/ml)和19周(24.56±9.65 U/ml)升高(P0.05),在0、8、12周無(wú)明顯改變(P0.05)。2.2大鼠肝勻漿中ADH、ALDH活性與對(duì)照組大鼠肝勻漿中ADH活性(3.43±1.07 U/mg prot)相比,大鼠肝勻漿ADH活性在模型A組(4.50±1.05 U/mg prot)、B(4.77±2.11 U/mg prot)組均升高(P0.05);對(duì)照組大鼠肝勻漿中ALDH活性為9.96±1.52 U/g prot,A、B模型組大鼠肝勻漿中ALDH活性分別為46.02±14.10 U/g prot和30.33±9.80U/g prot,較對(duì)照組顯著升高(P0.001),與模型A組相比,模型B組大鼠肝勻漿中ALDH活性下降34.1%(P0.001)。3大鼠ADH、ALDH和肝臟損傷相關(guān)指標(biāo)的關(guān)系3.1大鼠ADH基礎(chǔ)活性和肝臟損傷相關(guān)指標(biāo)的關(guān)系模型A組大鼠第0周血漿中ADH基礎(chǔ)活性和12周測(cè)定的4個(gè)肝臟損傷相關(guān)指標(biāo)有相關(guān)性(masson面積%,Ki67+%,PCNA+%,GST-p平均光密度值),相關(guān)系數(shù)分別為0.453、0.512、0.457、0.450(P0.05)。而模型B組大鼠血漿中ADH基礎(chǔ)活性和19周測(cè)定的肝臟損傷相關(guān)指標(biāo)不存在相關(guān)性(P0.05)。3.2大鼠肝勻漿中ADH、ALDH活性和和肝臟損傷相關(guān)指標(biāo)的關(guān)系模型B組大鼠肝勻漿中ADH活性和19周測(cè)定的5個(gè)肝臟損傷相關(guān)指標(biāo)(肝重/體重,結(jié)節(jié)數(shù)量,結(jié)節(jié)最大直徑,結(jié)節(jié)累積直徑,Ki67+%)有相關(guān)性,相關(guān)系數(shù)分別為0.567、0.499、0.438、0.626、0.506(P0.05)。模型B組肝勻漿中ADH/ALDH和19周測(cè)定的4個(gè)肝臟損傷相關(guān)指標(biāo)(肝重/體重,結(jié)節(jié)數(shù)量,結(jié)節(jié)最大直徑,結(jié)節(jié)累積直徑)存在相關(guān)性,相關(guān)系數(shù)分別為0.720、0.476、0.522、0.755(P0.01)。結(jié)論1.模型組大鼠血漿中ADH活性在16周和19周較對(duì)照組升高,肝勻漿中ADH活性在12周和19周較對(duì)照組升高。2.12周和19周DEN誘導(dǎo)的肝癌模型大鼠肝勻漿中ALDH活性較對(duì)照組升高。3.大鼠血漿中ADH基礎(chǔ)活性升高是肝纖維化的易感因素。
[Abstract]:Hepatocellular carcinoma (liver cancer) is the most common threat to human health of liver malignant tumor. Hepatitis cirrhosis hepatocellular carcinoma, the development model is supported. Liver fibrosis is the common pathological features of chronic liver disease, and chronic liver disease progression to cirrhosis stage. Alcohol dehydrogenase (alcohol dehydrogenases, ADH) and aldehyde dehydrogenase (aldehyde dehydrogenases, ALDH.ADH) mainly exists in the liver cells is mainly involved in the metabolism of ethanol, ethanol generated by the oxidation of acetaldehyde, acetaldehyde with liver toxicity, can induce cancer. At the same time, ADH is also involved in the metabolism of retinol, retinol and its metabolites in hepatitis and liver fibrosis, recent results show that ADH deficiency can inhibit liver fibrosis. According to reports, compared with the normal tissues adjacent to cancer. The activity of ADHI in liver cancer tissue increased. The activity of ADH in serum of patients with hepatocellular carcinoma compared with healthy individuals also increased. The room now During the results show that, compared with normal liver tissue, total ADH of hepatocellular carcinoma patients with liver fibrosis ADHI, ADHII activity were significantly increased, while the total ALDH and ALDH2 activity did not change. The results show that the increase of ADH activity and liver fibrosis or liver cancer may exist, but the activity of ADH in various stages of the development of HCC serum and the organization is how to change, increase the activity of ADH is the result of liver injury or cause the increase if there is further research on the susceptibility to liver injury. This study selected two diethylnitrosamine (diethylnitrosamine, DEN) of rat liver cancer model preparation as the research object, the determination of rats in different stage ADH activity induced by the correlation analysis of ADH based activity and liver injury related indicators in rat plasma, and analysis of liver tissue ADH, indicators of the correlation between ALDH activity and liver injury, in order to explore ADH 鍦ㄨ倽綰ょ淮鍖栫敋鑷寵倽鐧屽彂灞曚腑鎵，

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