發(fā)布時間：2018-02-27 19:05

  本文關(guān)鍵詞： 人參皂苷Rg1 細(xì)胞凋亡 內(nèi)質(zhì)網(wǎng)應(yīng)激 肝衰竭　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:急性肝衰竭病情危重、病死率極高,目前仍是臨床危重癥,除了人工肝替代治療、肝移植外,尚缺乏臨床治療特效藥物,因此探究臨床治療藥物非常必要。課題組前期研究發(fā)現(xiàn)人參皂苷Rg1(G-Rg1)對小鼠急性肝衰竭模型具有預(yù)防效果,且其可能與抗肝細(xì)胞凋亡和抗炎作用有關(guān),但G-Rg1的具體作用機制、靶點仍不清楚,需要進一步研究。因此擬建立四氯化碳(CCl4)誘導(dǎo)的急性肝衰竭小鼠模型,進一步研究G-Rg1對急性肝衰竭模型的作用機制,驗證G-Rg1對ALF小鼠模型的有效性,初步探討G-Rg1對CCl4誘導(dǎo)的ALF小鼠模型抗肝細(xì)胞凋亡的影響及相關(guān)機制。方法:采用隨機數(shù)字法將40只健康成年C57/BL雄性小鼠分為生理鹽水對照(NS)組,G-Rg1空白對照(G-Rg1)組,CCl4模型(CCl4)組,G-Rg1預(yù)防治療(CCl4+G-Rg1)組。建模12h后采集各組小鼠血清和肝臟組織,用試劑盒法檢測血清丙氨酸轉(zhuǎn)氨酶(alanine transaminase,ALT)、天冬氨酸轉(zhuǎn)氨酶(aspartate aminotransferase,AST)、總膽紅素(total bilirubin,Tbil)水平;HE染色評價肝臟組織病理學(xué)改變;定量PCR法檢測肝臟葡萄糖調(diào)節(jié)蛋白78(GRP78)、C/EBP家族同源蛋白(CHOP)的表達(dá)量;Western blot檢測GRP78、CHOP、半胱氨酸蛋白酶12(caspase12)、半胱氨酸蛋白酶3(caspase3)的表達(dá)情況;免疫組織化學(xué)法分析GRP78、caspase3的表達(dá)情況;TUNEL法檢測C57/BL小鼠肝臟組織細(xì)胞凋亡情況。結(jié)果:1.CCl4+G-Rg1組血清ALT、AST、TBil水平低于CCl4組,差異具有統(tǒng)計學(xué)意義(P0.05)。NS組、G-Rg1組、CCl4組、CCl4+G-Rg1組ALT值分別為(50.12±9.25)U/L、(40.48±6.38)U/L、(980.66±110.29)U/L、(691.30±108.06)U/L;AST值分別為(9.69±2.78)U/L、(9.40±3.84)U/L、(319.44±89.32)U/L、(195.40±15.41)U/L;TBil值分別為(0.46±0.13)mg/d L、(0.48±0.08)mg/d L、(1.56±0.12)mg/d L、(1.09±0.11)mg/d L。2.模型構(gòu)建完成后HE染色示CCl4組肝臟出現(xiàn)明顯變性壞死并可見肝細(xì)胞壞死灶,呈典型的CCl4所致肝細(xì)胞病理表現(xiàn)。經(jīng)G-Rg1預(yù)防治療后的CCl4+G-Rg1組肝臟組織變性壞死程度較CCl4組明顯減輕。3.CCl4+G-Rg1組GRP78、CHOP的m RNA相對表達(dá)量均較CCl4組下降,差異具有統(tǒng)計學(xué)意義(F=34.4、F=44.1,P0.05)。4.Western bolt結(jié)果顯示CCl4+G-Rg1組與CCl4組相比,caspase3、GRP78、caspase12、CHOP蛋白相對表達(dá)量均有不同程度降低,差異均有統(tǒng)計學(xué)意義(F=270.58、F=34.73、F=92.38、F=31.63,P0.05)。5.免疫組織化學(xué)法檢測GRP78、caspase3表達(dá)情況,光鏡下觀察結(jié)果顯示CCl4組較NS組胞漿內(nèi)棕黃色顆粒明顯增多。CCl4+G-Rg1組較CCl4組胞漿內(nèi)棕色顆粒減少。6.TUNEL結(jié)果顯示CCl4+G-Rg1組棕色顆粒物質(zhì)較CCl4組明顯減少(F=330.9,P0.05),提示G-Rg1預(yù)防治療可以減輕CCl4引起的肝細(xì)胞凋亡。結(jié)論:G-Rg1預(yù)防用藥可抑制CCl4誘導(dǎo)的小鼠急性肝衰竭的炎癥反應(yīng),同時可減輕肝細(xì)胞壞死和凋亡。其作用機制可能通過抑制內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)信號分子,改善肝細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激進而減輕肝細(xì)胞凋亡。結(jié)合課題組之前的研究結(jié)果提示G-Rg1可能通過多個作用靶點抑制肝臟炎癥反應(yīng)和肝細(xì)胞凋亡,實現(xiàn)保護肝臟功能作用。
[Abstract]:Objective: acute liver failure (ARF) is in critical condition and has a high mortality rate. It is still a clinical critical disease. In addition to artificial liver replacement therapy and liver transplantation, there is still a lack of effective drugs for the treatment of acute liver failure. Therefore, it is very necessary to explore the clinical therapeutic drugs. Our previous study found that ginsenoside Rg1 G-Rg1) has a preventive effect on acute liver failure in mice, and it may be related to the anti-hepatocyte apoptosis and anti-inflammatory effect, but the specific mechanism of G-Rg1. The target point is still unclear and needs further study. Therefore, we intend to establish an acute liver failure model induced by CCl4), to further study the mechanism of G-Rg1 on acute liver failure model, and to verify the effectiveness of G-Rg1 on ALF mouse model. To investigate the effect of G-Rg1 on anti-hepatocyte apoptosis in ALF mice induced by CCl4 and its related mechanism. Methods: 40 healthy adult C57rBL male mice were randomly divided into normal saline control group and G-Rg1 blank control group. The serum and liver tissues of each group were collected after 12 hours of modeling. Serum alanine transaminase (alt), aspartate aminotransferase (AST), total bilirubin (Tbilirubin) were detected by Kit method. The expression of Glucose-regulated protein 78 (GRP78) was detected by quantitative PCR, and the expression of GRP78, caspase12 and cysteine protease 3caspase3 was detected by Western blot. Immunohistochemical method was used to analyze the expression of GRP78 caspase3 and Tunel method was used to detect apoptosis in liver tissue of C57 / BL mice. Results 1. The level of serum alt ASTTBil in CCl4 G-Rg1 group was lower than that in CCl4 group. The ALT values of G-Rg1 group in CCl4 group were 50.12 鹵9.25 UL / L = 40.48 鹵6.38 U / L + 980.66 鹵110.29 U / L = 9.69 鹵108.06 U / L = 9.69 鹵2.78 U / L = 9.69 鹵2.78 渭 L / L 9.40 鹵3.84 渭 L / L ~ 319.44 鹵89.32 U / L / L ~ (195.40 鹵15.41) U / L / L = 0.46 鹵0.13 mg / d / L, respectively. The liver necrosis was observed after liver necrosis was observed in the liver necrosis group (P < 0.05 鹵0.12 mg / L 1.56 鹵0.12 mg / L 1.56 鹵0.12 mg / L 1.11 mg / d). The degree of degeneration and necrosis of liver tissue in CCl4 G-Rg1 group was significantly decreased than that in CCl4 group. 3. The relative expression of m RNA in GRP78 chop in CCl4 G-Rg1 group was lower than that in CCl4 group. The results of Western bolt showed that the relative expression of caspase3, GRP78, caspase12CHOP protein in the CCl4 G-Rg1 group was lower than that in the CCl4 group, and the difference was statistically significant in F270.58F34.73F74.73P0.05P0.05. the expression of GRP78caspase3 was detected by immunohistochemical method. The results of light microscopy showed that the brown granules in cytoplasm of CCl4 group were significantly higher than those of NS group. The brown granules in cytoplasm of CCl4 G-Rg1 group were less than those of CCl4 group. 6. Tunel results showed that the brown granule substance of CCl4 G-Rg1 group was significantly lower than that of CCl4 group, suggesting that G-Rg1 preventive therapy was possible. Conclusion the prophylaxis of CCl4 can inhibit the inflammatory response induced by CCl4 in mice with acute liver failure. At the same time, it can reduce hepatocyte necrosis and apoptosis. Its mechanism may be by inhibiting endoplasmic reticulum stress-related signaling molecules, In combination with previous research results, G-Rg1 may protect liver function by inhibiting liver inflammation and hepatocyte apoptosis through multiple targets.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.3;R-332

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