【摘要】：目的在細胞水平研究丙基硫氧嘧啶(PTU)對T4干預下人Hep G2細胞(模擬在甲亢環(huán)境下)的損傷機制,并探討異甘草酸鎂(Mg IG)對丙基硫氧嘧啶誘導的人Hep G2細胞損傷的保護作用。方法1、體外培養(yǎng)Hep G2細胞于100n M濃度的T4干預環(huán)境中,在含有PTU 0、75、150、300、600、1200、2400μg/ml一系列濃度培養(yǎng)基分別孵育不同的時間,利用過氧化氫(H2O2)體外誘導肝細胞損傷作為陽性對照,采用MTT比色法及LDH法檢測細胞增殖及細胞毒性情況,并測定谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的活性,得到最佳PTU誘導Hep G2細胞損傷濃度,建立PTU誘導的體外甲亢肝細胞損傷模型。2、將Hep G2細胞分成對照組、陽性對照組、PTU損傷組、Mg IG保護組,Hep G2細胞貼壁后,陽性對照組用H2O2誘導損傷,PTU損傷組和保護組則以PTU最佳損傷濃度預處理,8h后棄原培養(yǎng)液,Mg IG保護組加入5.0mg/ml Mg IG培養(yǎng)液中孵育24小時,其他組別補充等體積培養(yǎng)液。采用生化分析儀檢測天冬氨酸氨基轉(zhuǎn)移酶(AST)和丙氨酸氨基轉(zhuǎn)移酶(ALT)活性,試劑盒測定細胞胞漿中超氧化物歧化酶(SOD)的活性和谷胱甘肽(GSH)、丙二醛(MDA)的含量,探討異甘草酸鎂對PTU致肝損傷的保護作用。3、以PTU損傷模型作為研究對象,利用免疫印記法(Western blot)檢測細胞中凋亡相關蛋白Bax蛋白和Bcl-2蛋白的表達,c-Jun氨基末端激酶(JNK)、P38絲裂原活化蛋白激酶(P38)及其磷酸化形式蛋白的表達。結(jié)果1、PTU對Hep G2細胞的損傷作用隨劑量的增加和時間的增長而增強,在PTU濃度為600μg/ml,作用時間8h時對Hep G2細胞的抑制率為55.35±0.45%,死亡率為49.16±0.49%,培養(yǎng)液上清中ALT和AST水平較正常組明顯升高(P0.05)。2、異甘草酸鎂預處理后,與損傷組相比,異甘草酸鎂能顯著提高細胞生存率;降低Hep G-2細胞培養(yǎng)液上清中ALT和AST水平(P0.05),;降低細胞內(nèi)MDA水平(P0.05),同時增高細胞內(nèi)SOD的活性和GSH的含量(P0.05)。3、Western blot結(jié)果顯示,PTU能升高Hep G-2細胞JNK、P38及其磷酸化蛋白的表達,與損傷組相比,異甘草酸鎂保護組中的Bax蛋白表達降低,Bcl-2蛋白表達顯著升高(P0.05)。結(jié)論1.建立PTU致甲亢Hep G2細胞損傷模型的最佳干預濃度和時間分別為600μg/ml,8h;2.PTU致肝損傷的機制可能與JNK、p38MAPK通路的激活有關;3.異甘草酸鎂對PTU致Hep G2細胞損傷具有明顯的保護作用。
[Abstract]:Objective to study the damage mechanism of propylthiouracil (PTU) on human Hep G2 cells (simulated in hyperthyroidism) induced by T4 at the cellular level. To investigate the protective effect of magnesium isoglycyrrhizinate (Mg IG) on human Hep G2 cells induced by propyl thiouracil. Methods 1. Hep G2 cells cultured in vitro were incubated with T4 at 100nM concentration for different time on a series of culture medium containing PTU 075, 150, 300, 200 and 2 400 渭 g/ml. The hepatocyte injury induced by hydrogen peroxide (H2O2) in vitro was used as positive control. The cell proliferation and cytotoxicity were detected by MTT colorimetric assay and LDH assay, and the activities of (ALT) and (AST) were determined. The optimal concentration of PTU induced Hep G2 injury was obtained. PTU induced hyperthyroidism hepatocyte injury model was established in vitro. 2. Hep G2 cells were divided into three groups: control group, positive control group, Mg IG protection group, Hep G2 cell adhesion group, H2O2 induced injury in positive control group. The PTU injury group and the protection group were pretreated with the optimal concentration of PTU. After 8 hours, the, Mg IG group was incubated in the 5.0mg/ml Mg IG medium for 24 hours, and the other groups were supplemented with the same volume culture medium. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected by biochemical analyzer. The activities of superoxide dismutase (SOD) and glutathione (GSH), (GSH),) in the cytoplasm of cells were measured by kit. The content of malondialdehyde (MDA) and the protective effect of magnesium isoglycyrrhizinate on liver injury induced by PTU. 3. PTU damage model was used as the research object. The expression of apoptosis-related protein (Bax) and Bcl-2 protein and the expression of (JNK), P38 mitogen-activated protein kinase (P38) and its phosphorylated form protein (P38) were detected by (Western blot). Results 1the damage of Hep G2 cells was increased with the increase of dose and time. The inhibition rate of Hep G2 cells was 55.35 鹵0.45 and the mortality was 49.16 鹵0.49 when the concentration of PTU was 600 渭 g / ml and the exposure time was 8 h. The levels of ALT and AST in the supernatant of culture medium were significantly higher than those in the normal group (P0.05). After pretreatment with magnesium isoglycyrrhizinate, magnesium isoglycyrrhizinate could significantly improve the cell survival rate compared with the injured group. Decrease the levels of ALT and AST in the supernatant of Hep G-2 cells (P0.05),;) The results of Western blot showed that PTU could increase the expression of JNK,P38 and its phosphorylated protein in Hep G-2 cells. The expression of Bax protein decreased and the expression of Bcl-2 protein increased significantly in magnesium isoglycyrrhizinate group (P0.05). Conclusion 1. The optimal intervention concentration and time of PTU induced hyperthyroidism Hep G2 cell injury model were 600 渭 g / ml ~ (8) h ~ (-1) respectively. 2. The mechanism of PTU-induced liver injury may be related to the activation of JNK,p38MAPK pathway. 3. Magnesium isoglycyrrhizinate has obvious protective effect on Hep G2 cell injury induced by PTU.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R581.1

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