甘丙肽在小鼠非酒精性脂肪性肝炎发生发展中的作用初探

发布时间：2018-04-20 16:14

本文选题：甘丙肽 + 非酒精性脂肪性肝病　；参考：《上海交通大学》2014年硕士论文

【摘要】：背景与目的：随着人们生活水平的提高以及生活方式的改变，非酒精性脂肪性肝病(Nonalcoholic fatty liver disease, NAFLD)已逐渐成为21世纪全球重要的公共健康问题之一，其疾病谱主要包括单纯性脂肪肝(Simple fatty liver, SFL)、非酒精性脂肪性肝炎(Nonalcoholicsteatohepatitis, NASH)及相关肝纤维化和肝硬化。SFL可进展为NASH，更可在NASH基础上进一步发展为肝纤维化甚至肝硬化。已知炎症反应是SFL发展为NASH的关键限速步骤，而瘦素抵抗加速NASH的发生与进展。作为体内天然存在的能量代谢调控因子，甘丙肽许多重要生物学功能与瘦素相互拮抗，此外，，甘丙肽在机体炎症反应调控中也起一定作用。前期研究发现，在脂肪肝形成早期、SFL和NASH阶段，NAFLD动物模型的外周血甘丙肽水平存在动态变化，综上推测甘丙肽可能对NASH的发生发展有一定的影响。本研究将观察甘丙肽对NASH发生发展的影响，为非酒精性脂肪性肝病提供治疗靶点。方法：40只7～8周龄健康雄性C57BL/6小鼠经普通饲料适应性喂养10天后，随机分为正常对照组、模型组和干预组。正常对照组饲以普通饲料，模型组与干预组持续饲予高脂高胆固醇饲料，自始每周记录小鼠体重，每2周记录饲料消耗。饮食处理满8周后，干预组予以皮下注射甘丙肽，剂量8ug/100g体重，每天1次；正常对照组和模型组予以皮下注射等量磷酸盐缓冲生理盐水(Phosphate-Buffered Saline，PBS)，每天1次。饮食处理至第13周末实验结束，隔夜禁食后次日眼球摘除取血，颈椎脱臼处死动物。称量肝脏湿重、附睾脂肪重量；留取肝脏组织分别行苏木素-伊红(Hematoxylin and eosin，HE)染色评估肝脏组织病理学改变（脂肪性变与炎症程度）、免疫组织化学染色检测CD68(+)细胞数量和分布以判断肝内Kupffer细胞分布及浸润程度；采用实时定量PCR (Real-time PCR)法检测肝脏组织单核细胞趋化蛋白(Monocyte chemotactic protein，MCP)-1、 CD68、趋化因子受体(Chemokine receptor，CCR)-5、肿瘤坏死因子(Tumor necrosis factor,TNF)-、固醇调控元件结合蛋白(Sterol regulatory binding brotein，SREBP)-1c、过氧化物酶体增殖物激活受体(Peroxisomeproliferator-activated receptor, PPAR)-γ、瘦素长受体(Ob-Rb)的mRNA水平；采用自动生化仪测定各组小鼠血清生化指标，包括丙氨酸氨基转移酶(ALT)、甘油三酯、胆固醇、空腹血糖；采用酶联免疫吸附测定法(Enzyme-linked immunosorbent assay，ELISA)检测各组小鼠血清甘丙肽、胰岛素、瘦素和MCP-1水平。结果：与对照组相比，模型组与干预组小鼠体重及肝指数显著升高(p均0.01)。模型组肝组织病理学显示明显的肝细胞脂肪变及不同程度的炎症，NAFLD活动度积分(NAFLD activity score, NAS)和CD68(+)细胞计数均较对照组明显升高【(4.83±0.34) vs0；(3.62±4.51) vs(0.88±1.14)个/高倍视野，p0.01】；模型组血清ALT、甘油三脂、总胆固醇、MCP-1、胰岛素及瘦素水平均显著高于对照组水平【分别为(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】；模型组肝内CD68、MCP-1、CCR5、TNF-、SREBP-1c及PPAR-γ mRNA的水平亦均显著高于对照组(p均0.01)，而Ob-Rb mRNA水平则较对照组显著降低(p0.05)；给予甘丙肽干预后，干预组小鼠体重及肝指数与模型组无明显差异，但肝组织病理学检查示小叶内炎症积分和CD68阳性细胞计数为(0.27±0.12)和(0.84±1.23)个/高倍视野，均明显低于模型组水平【(0.92±0.19），p0.01；(3.62±4.51)个/高倍视野，p0.01】；干预组血清胰岛素水平和MCP-1蛋白水平也显著低于模型组【(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】，且与对照组水平相比无显著差异，但甘油三脂水平为(1.26±0.10) mmol/L，显著高于对照组和模型组【分别为(0.35±0.03) mmol/L和(0.66±0.09) mmol/L，p均0.01】；甘丙肽干预对瘦素水平无显著影响且仍高于对照组；干预组MCP-1、CCR5和TNF-和CD68mRNA水平显著低于模型组（p均0.05），且仅TNF-mRNA水平仍高于对照组水平；虽然干预组SREBP-1c和PPAR-γ表达仍高于正常对照组，但与模型组相比无显著差异，而干预组Ob-Rb表达水平显著高于模型组和对照组（p0.01）。结论：甘丙肽显著减轻高脂饮食诱导的小鼠NASH阶段的肝脏炎症反应，这一作用与减少肝内Kupffer细胞数量、减少炎症反应相关细胞因子的表达与分泌，以及上调瘦素受体改善瘦素抵抗有关，甘丙肽可能在抑制NASH炎症发应与疾病进展中起重要作用。
[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 ) - 纬 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 卤 7.38 ) vs ( 3.30 卤 0.34 ) U / L , p0.01 ; ( 0.66 卤 0.09 ) vs ( 0.35 卤 0.03 ) mmol / L , p0.01 ; ( 1.473 卤 0.09 ) vs ( 1.16 卤 0.09 ) ng / ml , p0.05 ; ( 19.42 卤 2.13 ) vs ( 6.27 卤 1.16 ) ng / ml , p0.01 ;
In the model group , the levels of CD68 , MCP - 1 , CCR5 , TNF - , IL - 1c and PPAR - 纬 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 卤 0.12 ) and ( 0.84 卤 1.23 ) / high - fold field of view in the interlobular inflammation score and ( 0.84 卤 1.23 ) / high - fold field of view , respectively , which were significantly lower than those in the model group ( 0.92 卤 0.19 ) , p0.01 ; ( 3.62 卤 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 卤 0.12 ) vs ( 1 . 47 卤 0.09 ) ng / ml , p < 0 . 05 ; ( 56 . 88 卤 2 . 03 ) vs ( 73.36 卤 3.35 ) pg / ml , p < 0.01 ) , but the levels of triglyceride were ( 1.26 卤 0.10 ) mmol / L , respectively ( 0.35 卤 0.03 ) mmol / L and ( 0.66 卤 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 .

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.1

【参考文献】