發(fā)布時間：2018-06-01 23:42

  本文選題：非酒精性脂肪性肝炎 + 性別間差異��； 參考：《吉林大學》2015年博士論文

【摘要】：第一部分MCD飲食誘導(dǎo)C57BL/6小鼠非酒精性脂肪性肝炎的性別間差異及其與MyD88依賴性IL-6信號通路的關(guān)系 目的：建立蛋氨酸-膽堿缺乏（methionine-choline-deficient，MCD）飲食誘導(dǎo)C57BL/6小鼠非酒精性脂肪性肝炎的動物模型，觀察此模型的性別間差異，探討該性別間差異與MyD88依賴性IL-6信號通路的關(guān)系。 方法：健康14周齡雄性C57BL/6小鼠26只，隨機分為2組：（1）雄性MCD組：予以MCD飲食；（2）雄性對照組：予以MCD對照飲食。健康14周齡雌性C57BL/6小鼠26只，隨機分為2組：（1）雌性MCD組：予以MCD飲食；（2）雌性對照組：予以MCD對照飲食。飼養(yǎng)4周后處死小鼠。自動生化分析儀檢測血清丙氨酸氨基轉(zhuǎn)移酶（alanine aminotransferase， ALT）、天門冬氨酸氨基轉(zhuǎn)移酶（aspartate aminotransferase，AST）、γ-谷氨酰轉(zhuǎn)肽酶（γ-Glutamyltransferase，γ-GT）水平。肝左葉部分肝組織行蘇木精—伊紅（hematoxylin-eosin,HE）、鋨酸染色觀察肝臟病理組織學改變，并進行非酒精性脂肪性肝病活動度積分（non-alcoholic fatty liver disease activity score，NAS）評定。同時應(yīng)用實時熒光定量RT-PCR方法測定肝組織髓樣分化因子88（myeloid differentiation primaryresponse gene88，MyD88）、白細胞介素-6（interleukin-6，IL-6）mRNA水平，應(yīng)用Western Blot蛋白印跡分析法測定肝組織MyD88、IL-6蛋白的表達水平。 結(jié)果：雄性MCD組和雌性MCD組小鼠肝小葉各區(qū)均可見肝細胞脂肪變性，為以大泡性為主的混合性脂肪變性，匯管區(qū)及小葉內(nèi)可見灶狀炎性細胞浸潤，以淋巴細胞浸潤為主。其中，雄性MCD組小鼠炎性細胞浸潤明顯,還可見肝細胞氣球樣變性。而雌性MCD組小鼠炎性細胞浸潤較少，未見肝細胞氣球樣變性。對照組小鼠肝組織形態(tài)正常，未見肝細胞脂肪變性和炎性細胞浸潤。 與各自對照組相比，雄性MCD組和雌性MCD組小鼠脂肪變性、炎癥評分、NAS積分及血清ALT、AST、γ-GT水平顯著升高（P＜0.05）。與雌性MCD組比較，雄性MCD組小鼠脂肪變性評分無顯著性差異（P＞0.05），但炎癥評分、NAS積分、血清ALT、AST、γ-GT水平顯著升高（P＜0.05），以ALT、AST升高為著。 與各自對照組相比，雄性MCD組和雌性MCD組小鼠肝組織MyD88、IL-6mRNA及其蛋白表達水平明顯升高（P＜0.05）。與雌性MCD組比較，雄性MCD組小鼠肝組織MyD88、IL-6mRNA及其蛋白表達水平明顯升高（P＜0.05）。 結(jié)論： MCD飲食誘導(dǎo)非酒精性脂肪性肝炎C57BL/6雄性小鼠肝臟病理損傷較雌性小鼠更為嚴重，炎性細胞浸潤及肝細胞氣球樣變性更加顯著，病理組織學評分顯著高于以同樣方法建立的雌性小鼠模型。MCD飲食誘導(dǎo)非酒精性脂肪性肝炎雄性小鼠的血清生化學改變較雌性小鼠更加顯著，其中ALT、AST升高較γ-GT更為明顯。MCD飲食誘導(dǎo)非酒精性脂肪性肝炎雄性小鼠肝組織MyD88、IL-6mRNA及蛋白表達水平均較雌性小鼠更加顯著，提示雄性及雌性小鼠模型在性別間的差異與MyD88依賴性IL-6信號通路的激活相關(guān)。 第二部分性別干預(yù)對MCD飲食誘導(dǎo)非酒精性脂肪性肝炎的影響及其與MyD88依賴性IL-6信號通路的關(guān)系 目的：建立MCD飲食誘導(dǎo)C57BL/6雄性小鼠非酒精性脂肪性肝炎的動物模型，通過手術(shù)去勢、手術(shù)去勢聯(lián)合補充外源性激素等方式，探討不同的性別干預(yù)方式對非酒精性脂肪性肝炎的影響，以及其與MyD88依賴性IL-6信號通路的關(guān)系。 方法：健康14周齡雄性C57BL/6小鼠65只，隨機分為5組：（1）雄性對照組：給予MCD對照飲食。（2）雄性MCD組：給予MCD飲食。（3）手術(shù)去勢MCD組：給予MCD飲食，并行手術(shù)切除睪丸。（4）手術(shù)去勢MCD+雄激素組：給予MCD飲食，并行手術(shù)切除睪丸及每日皮下注射丙酸睪酮5mg/kg。（5）手術(shù)去勢MCD+雌激素組：給予MCD飲食，并行手術(shù)切除睪丸及每日皮下注射苯甲酸雌二醇0.5mg/kg。飼養(yǎng)4周后處死小鼠。酶聯(lián)免疫吸附測定(enzyme-linkedimmunosorbent assay，ELISA)血清雌二醇、睪酮水平。自動生化分析儀測定血清ALT、AST、γ-GT水平。肝左葉部分肝組織行HE、鋨酸染色觀察肝臟病理組織學變化，并進行NAS積分評定。同時應(yīng)用實時熒光定量RT-PCR方法測定肝組織MyD88、IL-6mRNA水平，應(yīng)用Western Blot蛋白印跡分析法檢測肝組織MyD88、IL-6蛋白的表達水平。 結(jié)果：手術(shù)去勢可使小鼠血清睪酮水平顯著下降（P＜0.05）；補充外源性雄激素后，血清睪酮水平顯著升高（P＜0.05），恢復(fù)至未去勢水平；而補充外源性雌激素，可使小鼠血清雌二醇水平顯著升高（P＜0.05）。 與雄性MCD組相比，手術(shù)去勢MCD組小鼠肝細胞脂肪變性、炎性細胞浸潤及肝細胞氣球樣變性減輕，其NAS積分及血清ALT、AST水平也明顯降低（P＜0.05），血清γ-GT水平略降低（P＞0.05）；手術(shù)去勢MCD+雄激素組小鼠肝組織可見大量肝細胞脂肪變性，并有較多炎性細胞浸潤，偶可見肝細胞氣球樣變。與雄性MCD組、手術(shù)去勢MCD組相比，其NAS積分及血清ALT、AST、γ-GT水平均無顯著性差異（P＞0.05）；而手術(shù)去勢MCD+雌激素組小鼠肝組織中可見部分肝細胞脂肪變性，并有少量炎細胞浸潤，未見肝細胞氣球樣變。與雄性MCD組、手術(shù)去勢MCD組、手術(shù)去勢MCD+雄激素組相比，該組小鼠NAS積分和血清ALT、AST水平均明顯降低（P＜0.05）；但血清γ-GT僅在與雄性MCD組相比時具有顯著性差異（P＜0.05）。 與雄性MCD組比較，手術(shù)去勢MCD組小鼠肝組織MyD88、IL-6mRNA及其蛋白表達水平明顯降低（P＜0.05）。與雄性MCD組、手術(shù)去勢MCD組相比，手術(shù)去勢MCD+雄激素組小鼠肝組織MyD88、IL-6mRNA及其蛋白表達無明顯降低或升高（P＞0.05）。而手術(shù)去勢MCD+雌激素組小鼠肝組織MyD88、IL-6mRNA及其蛋白表達水平最低，并且與雄性MCD組、手術(shù)去勢MCD組、手術(shù)去勢MCD+雄激素組比較，均有顯著性差異（P＜0.05）。 結(jié)論：采用手術(shù)去勢或補充外源性雌激素進行性別干預(yù)后，可明顯改善MCD飲食誘導(dǎo)C57BL/6雄性小鼠非酒精性脂肪性肝炎的病理損傷，其中手術(shù)去勢聯(lián)合外源性雌激素組小鼠肝組織炎性細胞浸潤及脂肪變性最輕，NAS積分最低。手術(shù)去勢或補充外源性雌激素改善了肝臟病理損傷，也使血清ALT、AST水平較性別干預(yù)前顯著降低。單純手術(shù)干預(yù)對γ-GT影響不大，，但當手術(shù)聯(lián)合應(yīng)用外源性雌激素時，γ-GT也表現(xiàn)為顯著下降。采用手術(shù)去勢或補充外源性雌激素進行性別干預(yù)后，雄性C57BL/6小鼠肝組織MyD88、IL-6mRNA及蛋白的表達明顯下降，其中在手術(shù)去勢聯(lián)合應(yīng)用外源性雌激素組小鼠下降更為明顯。提示性別干預(yù)促進MCD飲食誘導(dǎo)C57BL/6雄性小鼠非酒精性脂肪性肝炎的恢復(fù)與其抑制MyD88依賴性IL-6信號通路相關(guān)。
[Abstract]:Part one: gender differences in MCD induced diet induced nonalcoholic steatohepatitis in C57BL/6 mice and their relationship with MyD88 dependent IL-6 signaling pathway
Objective: to establish an animal model of nonalcoholic steatohepatitis in C57BL/6 mice induced by methionine-choline-deficient (MCD) diet, and to observe the gender differences in this model and to explore the relationship between the gender differences and the MyD88 dependent IL-6 signaling pathway.
Methods: 26 healthy 14 week male C57BL/6 mice were randomly divided into 2 groups: (1) male MCD group: MCD diet, and (2) male control group: MCD control diet. 26 healthy 14 week old female C57BL/6 mice were randomly divided into 2 groups: (1) female MCD group: MCD diet; (2) female control group: MCD control diet. Feeding for 4 weeks after feeding. Dead mice. The serum alanine aminotransferase (alanine aminotransferase, ALT), aspartate aminotransferase (aspartate aminotransferase, AST), gamma glutamyl transaminopeptidase (gamma -Glutamyltransferase, gamma -GT) were detected by automatic biochemical analyzer. The liver left lobe of the liver was treated with hematoxylin Yi Hong (hematoxylin-eosin, HE), osmium acid staining. Liver histopathological changes were observed and the non-alcoholic fatty liver disease activity score, NAS was evaluated by the liver pathological changes, and the real time fluorescent quantitative RT-PCR method was used to determine the liver tissue myeloid differentiation factor 88 (myeloid differentiation primaryresponse gene88,), and interleukin (IL). Interleukin-6, IL-6) mRNA level, Western Blot protein blot analysis was used to detect the expression level of MyD88 and IL-6 protein in liver tissue.
Results: the hepatocyte fatty degeneration was found in all areas of the hepatic lobules in the male MCD group and the female MCD group, and it was a mixed fatty degeneration with large bullae. The infiltration of inflammatory cells in the tubular area and the lobule was visible, mainly with lymphocyte infiltration. Among them, the inflammatory cells in the male MCD group were obviously infiltrated, and the balloon like degeneration of the hepatocytes was also seen. In the female MCD group, the inflammatory cells in the mice were less infiltrated and no hepatocyte balloon like degeneration was found. The normal liver tissue of the control group was normal, and no fatty degeneration of hepatocytes and inflammatory cell infiltration were found in the control group.
Compared with the control group, the adipose degeneration, the score of inflammation, the score of NAS and the level of ALT, AST, and gamma -GT in the male MCD group and the female MCD group were significantly higher (P < 0.05). Compared with the female MCD group, there was no significant difference in the fat denaturation score of the male MCD group (P > 0.05), but the inflammatory score, NAS integral, serum ALT, and gamma levels were significantly higher than those in the female MCD group. 0.05), with ALT, AST rising.
Compared with the control group, the expression level of MyD88, IL-6mRNA and protein in the liver tissues of the male MCD group and the female MCD group increased significantly (P < 0.05). Compared with the female MCD group, the level of MyD88, IL-6mRNA and protein expression in the liver tissue of the male MCD group was significantly increased (P < 0.05).
Conclusion: MCD diet induced the liver pathological damage in the C57BL/6 male mice of nonalcoholic steatosis hepatitis more serious than that of female mice. Inflammatory cell infiltration and balloon like degeneration of liver cells were more significant. The histopathology score was significantly higher than that of the female mice model established by the same method of.MCD diet to induce nonalcoholic steatohepatitis. The serum biochemical changes in mice were more significant than those of female mice. The increase of ALT and AST was more obvious than that of gamma -GT in.MCD diet induced MyD88, IL-6mRNA and protein expression levels were more significant than that of female mice, suggesting the difference between sex and MyD88 dependent I in the male and female mice model. The activation of the L-6 signaling pathway is related.
The second part is the effect of gender intervention on MCD induced nonalcoholic steatohepatitis and its relationship with MyD88 dependent IL-6 signaling pathway.
Objective: to establish an animal model of nonalcoholic steatohepatitis in C57BL/6 male mice induced by MCD diet, and to explore the effects of different sex intervention on nonalcoholic steatohepatitis by surgical castration, operation castration combined with exogenous hormones and the relationship between the effect of different sex intervention on nonalcoholic steatohepatitis and the MyD88 dependent IL-6 signaling pathway.
Methods: 65 healthy 14 week male C57BL/6 mice were randomly divided into 5 groups: (1) the male control group was given the MCD control diet. (2) the male MCD group was given the MCD diet. (3) the operation castrated MCD group was given the MCD diet, and the testicles were excised in parallel. (4) the operation castrated MCD+ male IP group was given MCD diet, operation excision testis and subcutaneous daily subcutaneous Testosterone propionate 5mg/kg. (5) operation ovariectomized MCD+ estrogen group: MCD diet, parallel surgical resection of testicles and daily subcutaneous injection of estradiol benzoate 0.5mg/kg. to death mice. Enzyme linked immunosorbent assay (enzyme-linkedimmunosorbent assay, ELISA) serum estradiol, testosterone level. Automatic biochemical analyzer for serum determination of serum The level of ALT, AST and gamma -GT. The liver left lobe liver tissue was performed HE, the hepatic pathological changes were observed by osmium acid staining, and the NAS integral was evaluated. The MyD88, IL-6mRNA level of liver tissue was measured by real-time quantitative fluorescence quantitative RT-PCR method, and the expression level of liver tissue MyD88 and IL-6 protein was detected by the Western Blot Western blot analysis.
Results: the serum testosterone level of mice decreased significantly (P < 0.05). After supplementation of exogenous androgen, serum testosterone level increased significantly (P < 0.05) and recovered to the level of non castration, while supplementation of exogenous estrogen could significantly increase the level of serum estradiol in mice (P < 0.05).
Compared with the male MCD group, the hepatocyte fatty degeneration, inflammatory cell infiltration and hepatocyte balloon like degeneration were reduced in the operation castrated MCD group, and the NAS score and the serum ALT, AST level were also significantly decreased (P < 0.05), the serum level of gamma -GT decreased slightly (P > 0.05), and the liver tissue of the operation castrated MCD+ androgen group mice showed a large number of hepatocyte fatty degeneration, and There were many inflammatory cells infiltrating, and even the hepatocyte balloon like change. Compared with the male MCD group and the operation castrated MCD group, the NAS score and the serum ALT, AST, and gamma -GT levels were not significantly different (P > 0.05), while the liver tissue of the operation castrated MCD+ estrogen group of the surgical ovariectomized mice showed a partial hepatic steatosis, with a small number of inflammatory cells infiltrating and no liver fine. Compared with the male MCD group, the operation castration group MCD, the operation castrated MCD+ androgen group, the NAS scores and the serum ALT and AST levels of the mice were significantly decreased (P < 0.05), but the serum gamma -GT was significantly different from the male MCD group (P < 0.05).
Compared with the male MCD group, the expression level of MyD88, IL-6mRNA and protein in the liver tissue of the operation castrated MCD group was significantly lower (P < 0.05). Compared with the male MCD group and the operation castrated MCD group, the expression of MyD88, IL-6mRNA and protein expression in the hepatic tissue of the operation castrated MCD+ androgen group was not significantly reduced or increased (P > 0.05). The expression level of MyD88, IL-6mRNA and protein in the liver tissues of mice was the lowest, and was significantly different from that of the male MCD group, the operation castrated MCD group and the operation castrated MCD+ androgen group (P < 0.05).
Conclusion: the operation castration or supplementation of exogenous estrogen for the sex dry prognosis can obviously improve the pathological damage of non alcoholic steatohepatitis in the C57BL/6 male mice induced by MCD diet. The operation castration combined with exogenous estrogen group is the lightest of inflammatory cell infiltration and fatty degeneration in the liver tissue of the mice, the NAS score is the lowest. Supplementation of exogenous estrogen improved pathological injury of liver and decreased the level of serum ALT and AST significantly before the sex intervention. Simple operation intervention had little effect on gamma -GT, but when the operation combined with exogenous estrogen, gamma -GT also showed a significant decline. The prognosis of sex dry with surgical ovariectomy or supplementation of exogenous estrogen was performed in male C57. The expression of MyD88, IL-6mRNA and protein in the liver tissue of BL/6 mice decreased significantly, and the decrease in the combined use of exogenous estrogen group in the operation castration was more obvious. It suggested that the sex intervention to promote the recovery of non alcoholic steatohepatitis in C57BL/6 male mice induced by MCD diet was related to the inhibition of MyD88 dependent IL-6 signaling pathway.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R575.5

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