本文選題：甘丙肽 + 非酒精性脂肪性肝病��； 參考：《上海交通大學(xué)》2014年碩士論文

【摘要】：背景與目的：隨著人們生活水平的提高以及生活方式的改變，非酒精性脂肪性肝病(Nonalcoholic fatty liver disease, NAFLD)已逐漸成為21世紀(jì)全球重要的公共健康問題之一，其疾病譜主要包括單純性脂肪肝(Simple fatty liver, SFL)、非酒精性脂肪性肝炎(Nonalcoholicsteatohepatitis, NASH)及相關(guān)肝纖維化和肝硬化。SFL可進(jìn)展為NASH，更可在NASH基礎(chǔ)上進(jìn)一步發(fā)展為肝纖維化甚至肝硬化。已知炎癥反應(yīng)是SFL發(fā)展為NASH的關(guān)鍵限速步驟，而瘦素抵抗加速NASH的發(fā)生與進(jìn)展。作為體內(nèi)天然存在的能量代謝調(diào)控因子，甘丙肽許多重要生物學(xué)功能與瘦素相互拮抗，此外，，甘丙肽在機(jī)體炎癥反應(yīng)調(diào)控中也起一定作用。前期研究發(fā)現(xiàn)，在脂肪肝形成早期、SFL和NASH階段，NAFLD動物模型的外周血甘丙肽水平存在動態(tài)變化，綜上推測甘丙肽可能對NASH的發(fā)生發(fā)展有一定的影響。本研究將觀察甘丙肽對NASH發(fā)生發(fā)展的影響，為非酒精性脂肪性肝病提供治療靶點。 方法：40只7～8周齡健康雄性C57BL/6小鼠經(jīng)普通飼料適應(yīng)性喂養(yǎng)10天后，隨機(jī)分為正常對照組、模型組和干預(yù)組。正常對照組飼以普通飼料，模型組與干預(yù)組持續(xù)飼予高脂高膽固醇飼料，自始每周記錄小鼠體重，每2周記錄飼料消耗。飲食處理滿8周后，干預(yù)組予以皮下注射甘丙肽，劑量8ug/100g體重，每天1次；正常對照組和模型組予以皮下注射等量磷酸鹽緩沖生理鹽水(Phosphate-Buffered Saline，PBS)，每天1次。飲食處理至第13周末實驗結(jié)束，隔夜禁食后次日眼球摘除取血，頸椎脫臼處死動物。稱量肝臟濕重、附睪脂肪重量；留取肝臟組織分別行蘇木素-伊紅(Hematoxylin and eosin，HE)染色評估肝臟組織病理學(xué)改變（脂肪性變與炎癥程度）、免疫組織化學(xué)染色檢測CD68(+)細(xì)胞數(shù)量和分布以判斷肝內(nèi)Kupffer細(xì)胞分布及浸潤程度；采用實時定量PCR (Real-time PCR)法檢測肝臟組織單核細(xì)胞趨化蛋白(Monocyte chemotactic protein，MCP)-1、 CD68、趨化因子受體(Chemokine receptor，CCR)-5、腫瘤壞死因子(Tumor necrosis factor,TNF)-、固醇調(diào)控元件結(jié)合蛋白(Sterol regulatory binding brotein，SREBP)-1c、過氧化物酶體增殖物激活受體(Peroxisomeproliferator-activated receptor, PPAR)-γ、瘦素長受體(Ob-Rb)的mRNA水平；采用自動生化儀測定各組小鼠血清生化指標(biāo)，包括丙氨酸氨基轉(zhuǎn)移酶(ALT)、甘油三酯、膽固醇、空腹血糖；采用酶聯(lián)免疫吸附測定法(Enzyme-linked immunosorbent assay，ELISA)檢測各組小鼠血清甘丙肽、胰島素、瘦素和MCP-1水平。 結(jié)果：與對照組相比，模型組與干預(yù)組小鼠體重及肝指數(shù)顯著升高(p均0.01)。模型組肝組織病理學(xué)顯示明顯的肝細(xì)胞脂肪變及不同程度的炎癥，NAFLD活動度積分(NAFLD activity score, NAS)和CD68(+)細(xì)胞計數(shù)均較對照組明顯升高【(4.83±0.34) vs0；(3.62±4.51) vs(0.88±1.14)個/高倍視野，p0.01】；模型組血清ALT、甘油三脂、總膽固醇、MCP-1、胰島素及瘦素水平均顯著高于對照組水平【分別為(81.60±7.38) vs (3.30±0.34) U/L，p0.01；(0.66±0.09) vs (0.35±0.03)mmol/L，p0.05；(4.41±0.15) vs (3.30±0.34) mmol/L，p0.01；(73.36±3.35) vs (57.98±1.82) pg/ml，p0.05；(1.473±0.09) vs (1.16±0.09)ng/ml，p0.05；(19.42±2.13) vs (6.27±1.16) ng/ml，p0.01】；模型組肝內(nèi)CD68、MCP-1、CCR5、TNF-、SREBP-1c及PPAR-γ mRNA的水平亦均顯著高于對照組(p均0.01)，而Ob-Rb mRNA水平則較對照組顯著降低(p0.05)；給予甘丙肽干預(yù)后，干預(yù)組小鼠體重及肝指數(shù)與模型組無明顯差異，但肝組織病理學(xué)檢查示小葉內(nèi)炎癥積分和CD68陽性細(xì)胞計數(shù)為(0.27±0.12)和(0.84±1.23)個/高倍視野，均明顯低于模型組水平【(0.92±0.19），p0.01；(3.62±4.51)個/高倍視野，p0.01】；干預(yù)組血清胰島素水平和MCP-1蛋白水平也顯著低于模型組【(1.16±0.12) vs (1.47±0.09)ng/ml，p0.05；(56.88±2.03) vs (73.36±3.35) pg/ml，p0.01】，且與對照組水平相比無顯著差異，但甘油三脂水平為(1.26±0.10) mmol/L，顯著高于對照組和模型組【分別為(0.35±0.03) mmol/L和(0.66±0.09) mmol/L，p均0.01】；甘丙肽干預(yù)對瘦素水平無顯著影響且仍高于對照組；干預(yù)組MCP-1、CCR5和TNF-和CD68mRNA水平顯著低于模型組（p均0.05），且僅TNF-mRNA水平仍高于對照組水平；雖然干預(yù)組SREBP-1c和PPAR-γ表達(dá)仍高于正常對照組，但與模型組相比無顯著差異，而干預(yù)組Ob-Rb表達(dá)水平顯著高于模型組和對照組（p0.01）。 結(jié)論：甘丙肽顯著減輕高脂飲食誘導(dǎo)的小鼠NASH階段的肝臟炎癥反應(yīng)，這一作用與減少肝內(nèi)Kupffer細(xì)胞數(shù)量、減少炎癥反應(yīng)相關(guān)細(xì)胞因子的表達(dá)與分泌，以及上調(diào)瘦素受體改善瘦素抵抗有關(guān)，甘丙肽可能在抑制NASH炎癥發(fā)應(yīng)與疾病進(jìn)展中起重要作用。
[Abstract]:BACKGROUND & OBJECTIVE : With the improvement of people ' s living standard and the change of lifestyle , Nonalcoholic fatty liver disease ( NASH ) has gradually become one of the most important public health problems in the 21st century . The disease spectrum mainly includes simple fatty liver ( SFL ) , non - alcoholic steatohepatitis ( NASH ) and related liver fibrosis and cirrhosis .

Methods : Forty seven - eight week - old healthy male C57BL / 6 mice were randomly divided into normal control group , model group and intervention group .
The normal control group and the model group were given subcutaneous injection of phosphate buffered saline ( PBS ) once a day . The diet was treated to the end of the 13th weekend experiment . After overnight fasting , the eyeball was removed and blood was taken , and the animals were sacrificed . The weight of the liver was weighed and the fat weight of the appendix was weighed ;
Immunohistochemical staining was used to determine the number and distribution of CD68 ( + ) cells in liver tissues .
The mRNA levels of monocyte chemoattractant protein ( MCP ) - 1 , CD68 , chemokine receptor ( CCR ) -5 , tumor necrosis factor ( TNF ) - , Sterol regulatory binding brotein ( PPAR ) - 緯 and leptin receptor ( ob - Rb ) were detected by real - time PCR ( Real - time PCR ) .
The serum biochemical indexes , including alanine aminotransferase ( ALT ) , triglyceride , cholesterol and fasting plasma glucose , were determined by automatic biochemistry analyzer .
The levels of serum mannitol , insulin , leptin and MCP - 1 were measured by enzyme - linked immunosorbent assay ( ELISA ) .

Results : Compared with the control group , the body weight and liver index of the model group and the intervention group increased significantly ( p < 0 . 01 ) .
The levels of ALT , triglyceride , total cholesterol , MCP - 1 , insulin and leptin in model group were significantly higher than those in control group ( 81.60 鹵 7.38 ) vs ( 3.30 鹵 0.34 ) U / L , p0.01 ; ( 0.66 鹵 0.09 ) vs ( 0.35 鹵 0.03 ) mmol / L , p0.01 ; ( 1.473 鹵 0.09 ) vs ( 1.16 鹵 0.09 ) ng / ml , p0.05 ; ( 19.42 鹵 2.13 ) vs ( 6.27 鹵 1.16 ) ng / ml , p0.01 ;
In the model group , the levels of CD68 , MCP - 1 , CCR5 , TNF - , IL - 1c and PPAR - 緯 mRNA in the liver of the model group were significantly higher than those in the control group ( p < 0.01 ) , while the mRNA level of the ob - Rb mRNA was significantly lower than that of the control group ( p < 0.05 ) .
There was no significant difference in the body weight and liver index between the intervention group and the model group after the administration of the glycupropeptide , but the pathological examination of the liver showed that the inflammation score and the count of CD68 - positive cells were ( 0.27 鹵 0.12 ) and ( 0.84 鹵 1.23 ) / high - fold field of view in the interlobular inflammation score and ( 0.84 鹵 1.23 ) / high - fold field of view , respectively , which were significantly lower than those in the model group ( 0.92 鹵 0.19 ) , p0.01 ; ( 3.62 鹵 4.51 ) / high - fold field of view , p0.01 ;
The levels of serum insulin and MCP - 1 in the intervention group were significantly lower than those in the model group ( 1.16 鹵 0.12 ) vs ( 1 . 47 鹵 0.09 ) ng / ml , p < 0 . 05 ; ( 56 . 88 鹵 2 . 03 ) vs ( 73.36 鹵 3.35 ) pg / ml , p < 0.01 ) , but the levels of triglyceride were ( 1.26 鹵 0.10 ) mmol / L , respectively ( 0.35 鹵 0.03 ) mmol / L and ( 0.66 鹵 0.09 ) mmol / L , p - 0 . 01 respectively ;
The level of leptin had no significant effect on leptin level and was still higher than that of the control group .
The levels of MCP - 1 , CCR5 and TNF - and CD68 mRNA in the intervention group were significantly lower than those in the model group ( p < 0.05 ) , and the level of TNF - mRNA was still higher than that in the control group .
Although the expression levels in the intervention group were significantly higher than those in the normal control group , the expression level was significantly higher in the intervention group than in the model group and the control group ( p0.01 ) .

Conclusion : It can reduce the inflammatory response of the liver in the NASH stage induced by high fat diet significantly . This effect is related to the reduction of the number of ffer cells in the liver , the decrease of the expression and secretion of cytokines associated with inflammatory response , and the up - regulation of leptin receptor in leptin resistance , which may play an important role in inhibiting the progression of NASH inflammation .

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.1

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