【作者】 夏彦玲；

【导师】 李和平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2009， 博士

【摘要】 近年来各种肝病是严重威胁人类健康的主要疾病之一,因此,如何预防及治疗各类肝脏疾病已引起国内外学者的广泛重视。鹿茸是传统的滋补强壮剂,其化学成分和活性成分含量丰富,具有广泛的药理活性和医疗保健功能,鉴于此,本试验分别采用CCl₄、APAP、56°白酒、BCG+LPS建立小鼠化学性、药物性、酒精性和免疫性急性肝损伤模型,并在各型肝损伤试验中,将小鼠（20±1g）随机分为空白组、模型组、鹿茸醇提物大剂量组、鹿茸醇提物小剂量组、鹿茸粉组和联苯双酯组,通过制作肝组织的病理切片及电镜超薄切片,观察肝组织的病理改变及肝细胞的超微结构改变,研究鹿茸对小鼠肝损伤的保护作用,探讨其可能的肝损伤保护机制,为将其开发为抗肝损伤的新药提供理论依据。1.鹿茸对化学性肝损伤保护作用的显微及超微结构研究:（1）鹿茸能显著降低肝脏系数,减轻小鼠CCl₄急性肝损伤。各治疗组（鹿茸粉、鹿茸醇提物及联苯双酯组）与模型组（6.16±0.15%）比较肝脏系数均不同程度的降低,其中鹿茸醇提物大剂量组（5.25±0.09%）和小剂量组（5.21±0.12%）小鼠的肝脏系数明显降低,与模型组比较差异极显著（P＜0.01）。（2）HE切片显示:模型组小鼠肝组织可见有多处坏死灶并伴有炎性细胞浸润,肝小叶界限不清,肝细胞胞浆疏松,胞体肿胀,脂肪变性。各治疗组均有明显的改善,肝细胞的坏死、脂肪变性、细胞水肿及炎性浸润等病理改变减轻,鹿茸醇提物大剂量组效果好于其它治疗组,该组中大多数小鼠未见脂肪变性和坏死现象。统计分析表明:模型组与空白组比较差异极显著（p＜0.01）,说明造模成功,鹿茸醇提物大剂量组和阳性对照联苯双酯组与模型组比较病理改变明显减轻（p＜0.01）,鹿茸醇提物小剂量组和鹿茸粉组虽然病理改变也有所减轻,但与模型组比较差异不显著（p＞0.05）。（3）电镜超薄切片显示:模型组小鼠细胞膜破损,线粒体轮廓模糊,肿胀,嵴消失或断裂,内质网核糖体脱失,染色质凝集成块,细胞内有较多的脂滴,胆管微绒毛脱失,糖原颗粒减少。鹿茸制剂能改善上述染色质边集、内质网扩张、线粒体肿胀、细胞质膜受损等病理改变,表明鹿茸对脂质过氧化反应有拮抗作用,能有效地保护细胞膜及细胞器膜的完整性,并改善蛋白质、脂质及糖原在肝脏的代谢,其中鹿茸醇提物大剂量组细胞超微结构明显改善,已接近空白组水平,鹿茸粉及醇提物小剂量组细胞器的损伤也有所减轻。（4）鹿茸可使肝细胞中的脂滴减少,改善肝脏脂代谢功能,其中鹿茸醇提物大剂量组效果最好,偶见脂滴,已接近正常水平,醇提物小剂量组和联苯双酯组的效果相当,鹿茸粉的效果稍差。（5）鹿茸能减轻胆管及胆管微绒毛的损伤程度,恢复肝脏的分泌和排泄功能,各治疗组均有较好的作用。2.鹿茸对药物性肝损伤保护作用的显微及超微结构研究:（1）鹿茸能降低肝脏系数,减轻小鼠APAP肝损伤。鹿茸醇提物大剂量组的肝脏系数（5.42±0.09%）与模型组（5.91±0.15%）比较差异显著（P＜0.05）,醇提物小剂量组（5.62±0.07%）、鹿茸粉组（5.75±0.08%）和联苯双酯组（5.77±0.07%）的肝脏系数虽然有所减小,但与模型组比较差异不显著（P＞0.05）。（2）HE切片显示:模型组小鼠肝细胞结构模糊,肝细胞索排列紊乱,肝小叶结构不清,可见多处肝细胞坏死病灶,大部分细胞胞浆出现不同程度疏松,肝小叶中央及坏死区炎性细胞浸润明显。各治疗组中细胞坏死、水肿及炎性浸润等病理改变明显减轻,坏死灶明显减少。从病理分级情况的统计分析来看,各治疗组中,鹿茸醇提物大剂量组、小剂量组和阳性对照联苯双酯组与模型组比较病理改变均明显减轻,差异极显著（p＜0.01）,鹿茸粉组与模型组比较（p＜0.05）。（3）电镜超薄切片显示:模型组肝细胞出现液泡性坏死,粗面内质网扩张或裂解,线粒体嵴减少,电子密度降低,核膜皱缩,核基质变性,处于核固缩的前期,毛细胆管闭塞,消失,有少量脂滴,肝窦中可见中性粒细胞及成纤维细胞。各治疗组细胞质膜及线粒体、内质网等细胞器的病理改变均明显改善,总的说来鹿茸醇提物的效果好于鹿茸粉组,鹿茸醇提物大剂量组的作用效果与阳性对照联苯双酯组效果相当。（4）鹿茸对线粒体、内质网等细胞器损伤有较好的修复作用,从而抑制肝损伤引起的蛋白质合成功能的降低及脂代谢的紊乱,其中鹿茸醇提物大剂量组效果较其它治疗组效果明显,线粒体、内质网等细胞器基本恢复正常,鹿茸粉组效果最差。（5）鹿茸能改善肝脏的分泌和排泄功能,各治疗组效果均很好,其中鹿茸制剂组较阳性对照组效果明显。3.鹿茸对酒精性肝损伤保护作用的显微及超微结构研究:（1）鹿茸能降低肝脏系数,减轻小鼠酒精性肝损伤。鹿茸醇提物大剂量组（5.08±0.06%）、鹿茸粉组（5.37±0.09%）和联苯双酯组（5.18±0.07%）与模型组（5.88±0.09%）比较,均差异极显著（P＜0.01）,醇提物小剂量组（5.67±0.10%）与模型组比较差异显著（P＜0.05）。（2）HE切片显示:模型组小鼠肝小叶结构不清,肝细胞胞浆疏松,重者胞体肿大呈球形,胞浆透明,肝小叶散在点状坏死,主要见于中央静脉周围区,坏死灶及肝窦内可见炎性细胞浸润,轻度脂肪变性。各治疗组肝细胞的坏死、脂肪变性、细胞水肿及炎性浸润等病理改变明显改善,其中阳性对照联苯双酯组的效果略好于鹿茸醇提物大剂量组,鹿茸粉组与醇提物小剂量组效果稍差。从病理分级情况的统计分析来看,各治疗组中,鹿茸醇提物大剂量组和阳性对照联苯双酯组与模型组比较病理改变明显减轻,差异极显著（p＜0.01）,鹿茸醇提物小剂量组与模型组比较差异显著（p＜0.05）,鹿茸粉组虽然病理改变也有所减轻,但与模型组比较差异不显著（p＞0.05）。（3）电镜超薄切片显示:模型组肝细胞胞浆疏松,细胞器分散,线粒体严重肿胀,膜融合,嵴减少,有巨线粒体生成,基质密度降低,粗面内质网变化不明显,仅有部分粗面内质网轻微扩张,脱颗粒,核膜皱缩,染色质边集,胞质内有大量脂滴弥漫散在,有炎性细胞浸润,胆管闭锁,微绒毛融合,狄氏隙内有胶原纤维增生。各治疗组上述病理改变均有不同程度的减轻,其中鹿茸粉组相对来说效果略差一些,醇提物大剂量组、小剂量组和联苯双酯组效果均很好,醇提物大剂量组和联苯双酯组效果好于醇提物小剂量组,另外鹿茸能促进线粒体的增生,加快乙醇代谢。（4）鹿茸能明显抑制脂肪变性,减少肝细胞的脂滴数量,从而改善肝脏脂代谢功能,其中鹿茸醇提物大剂量组和联苯双酯效果最好,偶见脂肪滴,醇提物小剂量组和鹿茸粉的效果稍差。（5）鹿茸能减轻肝脏的胆汁淤滞,鹿茸醇提物大剂量组、小剂量组和联苯双酯组有效改善了胆管及胆管微绒毛的损伤程度,恢复肝脏的分泌和排泄功能,鹿茸粉组效果略差一些。4.鹿茸对免疫性肝损伤保护作用的显微及超微结构研究:（1）鹿茸能降低肝脏系数,减轻小鼠BCG和LPS诱导的免疫性肝损伤。鹿茸醇提物大剂量组（5.59±0.08%）和联苯双酯组（5.60±0.11%）与模型组（6.09±0.12%）比较,均差异极显著（P＜0.01）,醇提物小剂量组（5.77±0.09%）和鹿茸粉组（5.84±0.09%）与模型组比较差异不显著（P＞0.05）。（2）HE切片显示:模型组小鼠肝细胞索排列紊乱,存在大量的点状坏死,坏死区伴随出血和炎性细胞浸润,核明显大小不等,呈不同程度的固缩,嗜酸性增强,轻度脂肪变性,胞浆疏松,肝窦有淤血。鹿茸制剂能改善肝细胞的坏死、脂肪变性、细胞水肿及炎性浸润等病理改变。统计分析结果表明:各治疗组中,联苯双酯组和鹿茸醇提物大剂量组与模型组比较差异及显著（p＜<0.01）,鹿茸粉组与模型组比较差异显著（p＜0.05）,由于量效关系鹿茸醇提物小剂量组与模型组比较虽然有所改善,但差异不显著（p＞0.05）,而鹿茸醇提物大剂量组保肝效果与阳性对照联苯双酯组无明显区别。（3）电镜超薄切片显示:模型组小鼠肝细胞中脂滴增多,中性粒细胞和淋巴细胞增多,糖原增多,肝细胞微绒毛肿胀,肝窦缩小,窦内皮结构不清,胆汁淤滞,膜性结构融合,细胞膜及细胞器膜结构不清,核膜略有皱缩,线粒体肿胀,内质网减少,部分内质网轻度脱颗粒、肿胀。各治疗组的上述病理改变均有不同程度的改善,其中鹿茸粉组和醇提物小剂量组相对来说效果略差一些,醇提物大剂量组和联苯双酯组效果均很好,无明显的差别,醇提物大剂量组呈现轻微的病理改变。（4）鹿茸能改善肝脏脂代谢功能,减少TG在肝脏的堆积,其中鹿茸醇提物大剂量组脂滴极少。（5）鹿茸能改善肝脏的分泌和排泄功能,减轻胆汁淤滞的程度,鹿茸醇提物大剂量组效果最好,已接近空白组,其次是联苯双酯组,醇提物小剂量组和鹿茸粉组作用效果也很明显,但效果不及前两组。更多还原

【Abstract】 In recent years various liver diseases is one of the major diseases threating human health, so that prevention and treatment of various types of liver disease have attracted great attention from scholars at home and abroad.The velvet is the traditional tonic products with abundant chemical composition and active composition,which has extensive phamraeology activity and medical treatment and health care function.In order to observe protective effects of velvet on acute liver injury in mice and to explore its possible mechanisms,in the study we dealt the mice with CC14,APAP,Alcohol and BCG+LPS to establish the models of chemistry,drug, alcohol,and immune acute liver injury.Mice（20g±1g） were randomly divided into blank group,liver-injury model group,EAV（extract by alcohol from velvet,2.0g/kg） group and EAV（1.0g/kg） group,velvet powder group and bifendate group,and prepared paraffin section and ultrathin section of electron microscopy,observed pathological changes of liver tissue and liver cell ultrastructure.Our study could provid the theoretical basis for its development of new drugs of anti-liver injury.1.The study on microstructure and ultrastructure of velvet protective effects on chemical liver injury in mice:（1） Velvet could lower the mice’s liver coefficient obviously and reduce acute liver injury induced by CCl₄.Compared with the model group（6.16±0.15%） the effects of EAV groups（2.0g/kg,5.25±0.09%,1.0g/kg,5.21±0.12%） were the best（P＜0.01）,and other treatment groups also had better effects.（2） HE section showed that in model group there were obvious steatosis,focal necrosis with inflammatory collections,hepatielobule’s boundaries was unclear,celt swolled, the normal structure of cell disappeared,whereas the drug-treated groups relieved the necrosis,swelling,steatosis,inflammatory of hepatocytes.The effects of EAV（2.0g/kg） was better than other treatment groups,and there were no obvious necrosis and steatosis in the group.Statistical analysis showed that compared with blank group the CC14 group had significant difference（P＜0.01） and the mice acute hepatic injury experimential model was built very successfully.Compared with the model group EAV（2.0g/kg） and the positive control bifendate reduced the pathological changes significantly（p＜0.01）,although in EAV（1.0g/kg） and velvet powder groups the pathological changes had been reduced,the difference is not significant（p＞0.05）.（3） Ultrathin section of electron microscopy showed that in model group liver cell membrane damaged,mitochondria swollen and cristae disappeared or fractured,rough endoplasmic reticulum degranuled,chromatin condensated,fat droplets grown in number, microvilli of bile duct lost and glycogen reduced.Velvet could improve the condensation of chromatin,the expansion of mitochondria and RER,and other pathological changes,and improved the protein,fat and glycogen metabolism in the liver.The effects of EAV （2.0g/kg） was better than other groups,and had been near normal,the pathological changes in velvet powder and EAV low-dose groups also were reduced.（4） Velvet could reduce the lipid droplets and improve the liver fat metabolism.The effects of EAV（2.0g/kg） was better than other groups,near-normal liver architecture possessing higher hepatoprotective action,the effects of velvet powder was bad,the same effects occurred between BIF and EAV（1.0g/kg）.（5） Velvet could deincrease damage of the bile duct and microvilli,and restore the function of secretion and excretion of liver,every treatment group worked well.2.The study on microstructure and ultrastructure of velvet protective effects on drug-induced liver injury in mice:（） Velvet could lower the mice’s liver coefficient and reduce acute liver injury induced by APAP.Compared with the model group（5.91±0.15%） mice’s liver coefficient of EAV（2.0g/kg,5.42±0.09%） lowered significantly（P＜0.05）,although in other treatment groups mice’s liver coefficient had been reduced,the difference was not significant （p＞0.05）（2） HE section showed that in model group liver cell cord disordered,there were patches necrosis of liver cell with inflammatory collections around central vein and area of focal necrosis,hepatielobule’s structure was unclear,cell swollen,while the drug-treated groups relieved the necrosis、swelling、inflammatory of hepatocytes.Statistical analysis based on the degree of liver injury showed that compared with the model group EAV （2.0g/kg,1.0g/kg） and the positive control bifendate reduced the pathological changes significantly（p＜0.01）,the effects of velvet powder worked worst（p＜0.05）.（3） Ultrathin section of electron microscopy showed that in model group there was vacuole necrosis in hepatocytes,RER and mitochondria swollen,mitochondria’s cristae reduced and electron density lowerd,nuclear membrane shrunk,nuclear matrix degenerated at the early stage of karyopyknosis.Capillary bile duct closed up,small amount of lipid droplets、neutrophils and fibroblasts can be seen in liver cells.In treatment groups pathological changes of hepatocytes were improved significantly,generally for this respect,the effects of EAV were better than velvet powder,EAV（2.0g/kg） worked as well as BIF.（4） Velvet could repair damage of organelle such as mitochondria,endoplasmic reticulum and inhibit the reduction of protein synthesis and disorder of lipid metabolism caused by liver injury,from our study we could draw that the effects of EAV（2.0g/kg） were better than other treatment groups,and organelle was no obvious changes and near blank group,but the velvet powder was the worst.（5） Velvet could increase the function of liver’s secretion and excretion,every treatment group had a better role,and the mice treated with velvet agents had the lighter injury.3.The study on rnicrostructure and ultrastructure of velvet protective effects on the alcohol-induced liver injury:（1） Velvet could decreased the mice’s liver coefficient obviously and reduce acute liver injury induced by Alcohol.Compared with model group（5.88±0.09%） EAV group （2.0g/kg,5.08±0.06%）、velvet powder（5.37±0.09%） and BIF（5.18±0.07%） had significant differences（P＜0.01）,and EAV（1.0g/kg） was worse（P＜0.05）,.（2） HE section showed that in model group hepatielobule’s boundaries was unclear, cell swollen and the worst displayed bolloning generation,there were scattered punctate necrosis around central vein,fatty degeneration of hepatocytes and inflammatory around area of necrosis or in sinusoid;while the drug-treated groups relieved the necrosis,swelling, steatosis,inflammatory of hepatocytes.In term of it the effects of BIF was slightly superior to EAV（2.0g/kg）,and EAV（1.0g/kg） and velvet powder were a little worse.Statistical analysis showed that compared with the model group the effects of EAV（2.0g/kg） and BIF were better（p＜0.01）,EAV（1.0g/kg） was a little worse（p＜0.05）,and velvet powder was the worst（p＞0.05） but liver injury was relieved in every treatment group.（3） Ultrathin section of electron microscopy showed that in model group hepatic cells loose,organelle scattered,mitochondrial seriously swollen,membrane fusion,cristae reduced and giant mitochondria generated,rough endoplasmic reticulum did not change significantly with only slight expansion and degranulation.Nuclear membrane shrunk,chromatin marginated,there were a lot of fat droplets in liver cell and inflammatory,capillary bile duct closed up,there were collagen fibers hyperplasia in dieldrin gap.Treatment groups had relieved the above-mentioned pathological changes to different extents.For protecting liver’s injury,the effects of EAV（1.0g/kg） were better than the velvet powder but worse than the EAV （2.0g/kg） and BIF（worked as well as EAV（2.0g/kg））.In addition velvet promoted hyperplasia of mitochondria,to speed up the metabolism of ethanol.（4） Velvet could inhibit fatty degeneration and ruduced the number of fat droplets,thus improved liver function of lipid metabolism.The effects of EAV（2.0g/kg） and BIF were the best,while EAV（1.0g/kg） and velvet powder were a little worse.（5） Velvet could alleviate cholestasis of liver in every treatment group obviously,the effects of velvet powder was the less worse.EAV and BIF could be seen improve effectively the the damage of bile duct and microvilli to restore the secretion and excretion function of liver.4.The study on microstructure and ultrastructure of velvet protective effects on the immunological liver injury:（1） Velvet could decreased the mice’s liver coefficient obviously and reduce acute liver injury induced by BCG+LPS.Compared with model group（6.09±0.12%）,EAV（2.0g/kg, 5.59±0.08%） and BIF（5.60±0.11%） had significant differences（P＜0.01）,EAV （1.0g/kg,5.77±0.09%） and velvet powder（5.84±0.09%） was worse（P＞0.05）.（2） HE section showed that in model group liver cell cord disorders,there were the existence of a large number of punctate necrosis with inflammatory and hemorrhage,nuclear size were different and showed shrinkage of different degrees.Eosinophilic enhanced,and there were mild fatty degeneration,there was siltation of blood in sinusoid,while the drugtreated groups relieved the necrosis,swlling,steatosis,inflammatory of hepatocytes.The results of statistical analysis showed that compared with the model group the effects of BIF and EAV（2.0g/kg） were significant different（p＜0.01）,velvet powder（p＜0.05）,because of dose-effect relationship in EAV（1.0g/kg） the difference was not significant（p＞0.05）, meanwhile there was no difference between EAV（2.0g/kg） and BIF.（3） Ultrathin section of electron microscopy showed that in model group the number of small lipid droplets,glycogen,neutrophils and lymphocytes increased,microvilli of hepatocyte swollen,sinusoidal narrowed and sinus endothelial structure was unclear.The membrane structure of cell and organelle fused,mitochondrial swollen,the number of endoplasmic reticulum reduced,some endoplasmic reticulum had mild degranulation and swelling.Treatment groups had different relief of the above-mentioned pathological changes. The effects of EAV（2.0g/kg） and BIF surpassed that of EAV（1.0g/kg） and velvet powder,and EAV（2.0g/kg） group had been near blank group.（4） In terms of improving lipid metabolism velvet could inhibit fatty degeneration,and reduce accumulation of TG in the liver.The effects of EAV（2.0g/kg） was better than other treatment group and there were few fat droplets.（5） Velvet could increase the secretion and excretion function of liver,and alleviate cholestasis of liver,the every treatment group had a better role,and the mice treated with velvet agents had the lightst injury.EAV（2.0g/kg） were the best and close to the blank group, followed by the BIF group,the effects of EAV（1.0g/kg） and velvet powder was also evident,but less than the former two groups.更多还原