【摘要】：隨著藥物濫用的現(xiàn)象日益嚴(yán)重,藥物引起的肝臟問(wèn)題也逐漸增加,成為臨床上最常見(jiàn)的肝臟問(wèn)題之一。醋氨酚(APAP)是常用的解熱鎮(zhèn)痛藥,其造成的肝臟問(wèn)題在藥物性肝臟問(wèn)題中比例最高。醋氨酚誘導(dǎo)的肝損傷模型是研究藥物性肝損傷的經(jīng)典模型。谷胱甘肽S轉(zhuǎn)移酶A1(GSTA1)是機(jī)體的代謝酶系統(tǒng)之一,可保護(hù)細(xì)胞應(yīng)對(duì)氧化應(yīng)激,抵抗外來(lái)毒性物質(zhì)和活性氧的產(chǎn)生而保護(hù)機(jī)體。肝細(xì)胞核因子-1(HNF-1)被確定具有與幾種肝基因啟動(dòng)子相互作用的DNA結(jié)合活性,在肝特異性轉(zhuǎn)錄中起重要作用。因此,它在調(diào)節(jié)肝臟的生長(zhǎng)發(fā)育和代謝發(fā)揮著不可或缺的作用。本課題在APAP誘導(dǎo)的肝細(xì)胞損傷模型中,通過(guò)C2-神經(jīng)酰胺降低或奧替普拉升高HNF-1表達(dá)的兩種情況下對(duì)GSTA1表達(dá)的影響,確定HNF-1對(duì)GSTA1表達(dá)的調(diào)節(jié)作用,為明確GSTA1在藥物性肝細(xì)胞損傷中的重要作用,研究藥物性肝損傷提供體外試驗(yàn)依據(jù),并為深入研究其作用機(jī)制及保護(hù)肝損傷提供理論基礎(chǔ)。本試驗(yàn)采用系列濃度的醋氨酚作用于肝細(xì)胞,通過(guò)檢測(cè)培養(yǎng)上清液轉(zhuǎn)氨酶(ALT、AST)活性、肝細(xì)胞指標(biāo)(SOD、MDA、GSH、GSH-Px)水平來(lái)判斷肝細(xì)胞的損傷程度,優(yōu)化APAP作用濃度并復(fù)制肝細(xì)胞損傷模型。在此模型的基礎(chǔ)上,通過(guò)檢測(cè)轉(zhuǎn)氨酶活性來(lái)確定C2-神經(jīng)酰胺和奧替普拉的最適作用濃度。隨后,進(jìn)行HNF-1調(diào)節(jié)GSTA1表達(dá)的試驗(yàn)。本試驗(yàn)分為六組,即對(duì)照組、模型組、C2-神經(jīng)酰胺組(C2組)、C2-神經(jīng)酰胺+APAP組(C2+APAP組)、奧替普拉組(OL組)、奧替普拉+APAP組(OL+APAP組),應(yīng)用試劑盒檢測(cè)培養(yǎng)上清ALT、AST和肝細(xì)胞指標(biāo)SOD、GSH-Px、MDA和GSH;采用Real-time RT-PCR方法檢測(cè)肝細(xì)胞中HNF-1和GSTA1 m RNA的相對(duì)表達(dá),應(yīng)用Western blot方法測(cè)定肝細(xì)胞中HNF-1和GSTA1蛋白的相對(duì)表達(dá),并應(yīng)用試劑盒檢測(cè)培養(yǎng)上清中GSTA1的含量變化。通過(guò)以上研究,明確在APAP誘導(dǎo)的藥物性肝細(xì)胞損傷中,HNF-1對(duì)GSTA1表達(dá)是否具有調(diào)節(jié)作用。研究結(jié)果表明:1、采用系列濃度APAP作用10 h可對(duì)肝細(xì)胞產(chǎn)生不同程度的損傷。當(dāng)APAP濃度為15 m M時(shí),培養(yǎng)上清轉(zhuǎn)氨酶活性顯著升高(p0.05);肝細(xì)胞指標(biāo)(MDA、GSH、GSH-Px)水平均有極顯著性變化(p0.01),肝細(xì)胞SOD顯著降低(p0.05);細(xì)胞狀態(tài)不佳,出現(xiàn)變形,皺縮等,最終確定以15 m M APAP作為模型組濃度,并成功復(fù)制藥物性肝細(xì)胞損傷模型。2、通過(guò)最適作用濃度篩選試驗(yàn),確定C2-神經(jīng)酰胺以6μM濃度、奧替普拉以8μM濃度作用細(xì)胞。3、C2-神經(jīng)酰胺和奧替普拉作用于肝細(xì)胞損傷模型的結(jié)果顯示,與對(duì)照組比較,C2組和OL組培養(yǎng)上清液轉(zhuǎn)氨酶活性、肝細(xì)胞指標(biāo)均無(wú)顯著性變化,顯示出6μM的C2-神經(jīng)酰胺和8μM奧替普拉不會(huì)對(duì)肝細(xì)胞產(chǎn)生影響。與模型組比較,C2+APAP組中轉(zhuǎn)氨酶活性極顯著升高(p0.01),肝細(xì)胞指標(biāo)均呈極顯著變化(p0.01),顯示出C2-神經(jīng)酰胺會(huì)加劇APAP誘導(dǎo)的肝細(xì)胞損傷;OL+APAP組轉(zhuǎn)氨酶活性極顯著降低(p0.01),肝細(xì)胞指標(biāo)也均呈極顯著變化(p0.01),表明奧替普拉能減輕APAP誘導(dǎo)的肝細(xì)胞損傷。4、HNF-1的表達(dá)結(jié)果表明,與對(duì)照組比較,模型組HNF-1 m RNA和蛋白相對(duì)表達(dá)量均極顯著降低(p0.01),而C2組和OL組均無(wú)明顯變化;與模型組比較,C2+APAP組HNF-1的mRNA和蛋白相對(duì)表達(dá)水平極顯著降低(p0.01),而OL+APAP組極顯著升高(p0.01)。上述結(jié)果顯示出,在APAP誘導(dǎo)的肝細(xì)胞損傷中,C2-神經(jīng)酰胺降低了HNF-1的表達(dá),奧替普拉升高了HNF-1的表達(dá)。5、GSTA1的表達(dá)及含量測(cè)定的結(jié)果顯示,與對(duì)照組比較,模型組GSTA1 m RNA和蛋白相對(duì)表達(dá)量均極顯著降低(p0.01),培養(yǎng)上清液GSTA1含量極顯著升高(p0.01),而C2組和OL組均無(wú)顯著性變化;與模型組比較,C2+APAP組中GSTA1 m RNA和蛋白的相對(duì)表達(dá)均極顯著降低(p0.01),OL+APAP組均極顯著升高(p0.01);C2+APAP組培養(yǎng)上清液GSTA1含量極顯著增加(p0.01),OL+APAP組極顯著降低(p0.01)。上述結(jié)果表明,在APAP誘導(dǎo)的肝細(xì)胞損傷中,GSTA1在基因和蛋白水平上的表達(dá)趨勢(shì)均與HNF-1相一致。本研究成功復(fù)制APAP誘導(dǎo)的肝細(xì)胞損傷模型。確定C2-神經(jīng)酰胺和奧替普拉的最適作用濃度。在APAP誘導(dǎo)的肝細(xì)胞損傷中,C2-神經(jīng)酰胺可加重肝細(xì)胞損傷,奧替普拉可減輕肝細(xì)胞損傷。在肝細(xì)胞損傷的情況下,C2-神經(jīng)酰胺可抑制HNF-1的表達(dá),而奧替普拉可促進(jìn)HNF-1的表達(dá)。確定HNF-1對(duì)GSTA1表達(dá)的調(diào)節(jié)作用,且基因和蛋白表達(dá)水平的趨勢(shì)相同。HNF-1可通過(guò)調(diào)節(jié)GSTA1表達(dá)來(lái)保護(hù)肝細(xì)胞,但其機(jī)制尚需研究。
[Abstract]:Acetaminophen (APAP) is one of the most common antipyretic and analgesic drugs, which causes the highest proportion of liver problems in drug-induced liver injury. Acetaminophen-induced liver injury model is to study drug-induced liver injury. Classical model. Glutathione S-transferase A1 (GSTA1) is one of the metabolic enzymes in the body. It protects cells against oxidative stress and the production of toxic substances and reactive oxygen species. Hepatocyte nuclear factor-1 (HNF-1) has been identified as a DNA binding activity interacting with several liver gene promoters and is involved in liver-specific transcription. Therefore, it plays an indispensable role in regulating the growth, development and metabolism of the liver. In the APAP-induced hepatocyte injury model, through the reduction of C2-ceramide or the elevation of HNF-1 expression by otipra, we determined the regulatory effect of HNF-1 on GSTA1 expression, in order to clarify GSTA1 expression. TA1 plays an important role in drug-induced hepatocyte injury, and provides a theoretical basis for further study of its mechanism and protection against liver injury. In this study, a series of concentrations of acetaminophen were used to treat hepatocytes. The activity of ALT and AST in culture supernatant and hepatocyte index (SOD, M) were detected. DA, GSH, GSH-Px) levels were used to determine the degree of hepatocyte injury, optimize APAP concentration and replicate the hepatocyte injury model. On the basis of this model, the optimal concentration of C2-ceramide and otipra was determined by detecting transaminase activity. Model group, C2-ceramide group (C2 group), C2-ceramide+APAP group (C2+APAP group), Otipra group (OL group), Otipra+APAP group (OL+APAP group), using kits to detect ALT, AST and hepatocyte indicators SOD, GSH-Px, MDA and GSH; Real-time RT-PCR to detect the relative expression of HNF-1 and GSTA1 m RNA in hepatocytes, and Western RT-PCR to detect the expression of HNF-1 and GSTA1 m RNA in hepatocytes. Western blot was used to detect the relative expression of HNF-1 and GSTA1 proteins in hepatocytes, and a kit was used to detect the content of GSTA1 in culture supernatant. When the concentration of APAP was 15 m M, the activity of transaminase in the supernatant increased significantly (p0.05); the levels of hepatocyte markers (MDA, GSH, GSH-Px) changed significantly (p0.01); the SOD of hepatocytes decreased significantly (p0.05); the cells were in poor condition, deformed and shrunken. Finally, the concentration of 15 mMAP was determined as the model group. The model of drug-induced hepatocyte injury was successfully reproduced. 2. The optimal concentration of C2-ceramide was determined by screening test. Cell injury was induced by C2-ceramide at 6 mu M, cell injury was induced by otipra at 8 mu M, and transaminase activity in supernatant of C2 and OL groups was determined by C2-ceramide and otipra. Compared with the model group, the transaminase activity in C2 + APAP group was significantly increased (p0.01), and the hepatocyte index was significantly changed (p0.01), indicating that C2-ceramide could aggravate the hepatocyte injury induced by APAP. The activity of transaminase and hepatocyte index in L + APAP group were significantly decreased (p0.01), indicating that Otipra could alleviate APAP-induced hepatocyte injury. 4. The expression of HNF-1 m RNA and protein in model group were significantly decreased compared with control group (p0.01), but there was no significant change in C2 group and OL group. Compared with the model group, the expression of HNF-1 mRNA and protein in C2+APAP group was significantly decreased (p0.01), while that in OL+APAP group was significantly increased (p0.01). The results showed that the relative expression of GSTA1 m RNA and protein in model group was significantly lower than that in control group (p0.01), and the content of GSTA1 in culture supernatant was significantly higher (p0.01), but there was no significant change in C2 group and OL group. Compared with model group, the relative expression of GSTA1 m RNA and protein in C2 + APAP group was significantly lower (p0.01), and that in OL + APAP group was extremely high (p0.01). The results showed that the expression of GSTA1 at gene and protein levels was consistent with that of HNF-1 in APAP-induced hepatocyte injury. C2-ceramide can aggravate hepatocyte injury in APAP-induced hepatocyte injury, while otipra can alleviate hepatocyte injury. C2-ceramide can inhibit the expression of HNF-1 in the case of hepatocyte injury, while otipra can promote the expression of HNF-1. HNF-1 can protect hepatocytes by regulating the expression of GSTA1, but its mechanism needs to be studied.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 謝雅清;梁曉美;葉偉霞;;還原型谷胱甘肽的藥理作用與臨床應(yīng)用研究進(jìn)展[J];中國(guó)藥業(yè);2013年07期

2 喬玉成;;老齡大鼠腦組織SOD/MDA與NO/NOS的相關(guān)性研究[J];中國(guó)體育科技;2009年02期

3 章軼鋒;唐善虎;秦文玲;張巍;謝芳;;銅鋅超氧化物歧化酶的研究進(jìn)展[J];四川畜牧獸醫(yī);2008年01期

4 袁平戈;張大志;;還原型谷胱甘肽的作用機(jī)制及臨床應(yīng)用[J];藥品評(píng)價(jià);2006年05期

5 遲乃玉,張慶芳,劉長(zhǎng)江;SOD的化學(xué)特性及其應(yīng)用[J];沈陽(yáng)農(nóng)業(yè)大學(xué)學(xué)報(bào);1999年02期

相關(guān)博士學(xué)位論文 前2條

1 吳宇;藥物性肝損傷體外篩選模型和何首烏致肝損傷的初步研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

2 詹亦貝;5'-AMP對(duì)哺乳動(dòng)物急性肝損傷保護(hù)作用的研究[D];南京理工大學(xué);2014年

相關(guān)碩士學(xué)位論文 前4條

1 馬欣;醋氨酚誘導(dǎo)小鼠急性藥物性肝損傷中HNF-1調(diào)節(jié)GSTA1表達(dá)分析[D];東北農(nóng)業(yè)大學(xué);2016年

2 荊孝東;三肽模擬谷胱甘肽過(guò)氧化物酶及其生物學(xué)效應(yīng)研究[D];吉林大學(xué);2014年

3 劉穎姝;急性肝損傷早期診斷指標(biāo)GSTA1的研究及保肝藥物初篩[D];東北農(nóng)業(yè)大學(xué);2013年

4 鄧同興;神經(jīng)酰胺調(diào)節(jié)酒精誘導(dǎo)小鼠神經(jīng)細(xì)胞增殖的實(shí)驗(yàn)研究[D];鄭州大學(xué);2011年

，

本文編號(hào)：2246071