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板蓝根多糖对大鼠肝脏缺血—再灌注损伤的保护作用
【作者】 苏辉；
【作者基本信息】 扬州大学 ， 外科学， 2011， 硕士
【摘要】 1、目的通过构建大鼠自体原位肝移植(AOLT)模型,比较板蓝根多糖灌胃组大鼠与对照组大鼠自体原位肝移植术后早期肝脏病理改变、生化指标及TNF-α表达差异,研究板蓝根多糖对大鼠自体原位肝移植模型肝脏缺血-再灌注损伤的保护作用,分析其对肝移植大鼠肝脏功能保护作用的可能机制,为进一步探索肝移植提供实验基础。2、方法2.1清洁级SD纯系大鼠72只,雌雄不拘,随机分为3组：自体原位肝移植模型组(AOLT组n=24)；板蓝根多糖+自体原位肝移植组(RIPT组n=24)；假手术组(Sham组n=24)。两组自体原位肝移植大鼠均采用经门静脉灌注的70%自体肝移植模型,并加以技术改进施行大鼠自体原位肝移植手术,Sham组行开关腹及解剖肝周韧带及血管手术,但术中不行灌肝处理。2.2比较大鼠自体原位肝移植术后48h内的相关指标变化水平。各组大鼠均在术后1h、12h、24h及48h时间段各分别处死6只,经下腔静脉采血、分离血清,全自动生化仪检测ALT、AST等生化指标,ELISA法检测血清TNF-α,切取肝中叶作为标本制备切片,在光镜、透射电镜下观察肝细胞结构形态学改变、肝细胞线粒体及细胞器的损伤情况。3、结果3.1手术结束后,大鼠即刻苏醒,翻身,并能维持有效呼吸、心跳。经门静脉灌注,肝脏热缺血时间一般小于5秒,可以忽略。无肝期结束后,RIPT组肝脏复色均匀,色泽红润；AOLT组较RIPT组复色稍差,局部可见花斑样改变。3.2分析各组肝功能测定结果,AOLT组及RIPT组术后1h血清ALT和AST即明显升高,于术后1h、12h及24h均显著高于Sham组,差异有统计学意义(P<0.05);RIPT组随着术后时间的延长血清ALT和AST逐步回落,RIPT组血清ALT和AST在12h及24h时间点检测值显著低于对应时间段AOLT组检测值,差异有统计学意义(P<0.05)。3.3光镜下术后各组肝细胞的病理变化：RIPT组及AOLT组,在术后1h观察见肝细胞肿胀明显,肝小叶结构基本正常,其中AOLT组肝细胞之间的间隙变宽,肝血窦变窄较明显,内有淤积的红细胞并见血栓形成,周围出现炎症细胞浸润；Sham组肝细胞结构正常,无炎症细胞浸,肝组织无明显改变。术后12h,RIPT组肝细胞肿胀略重,肝组织无明显改变；AOLT组肝细胞水肿加剧,肝血窦明显变窄,肝小叶结构不清,内有淤积的红细胞和血栓,周围有炎症细胞浸润。术后24h, RIPT组肝细胞肿胀稍好转,肝血窦变宽；AOLT组,肝细胞肿胀进一步加重,肝血窦继续变窄,肝小叶结构显示不清。3.4观察透射电子显微镜下各组肝细胞超微结构可见：术后1h,RIPT组大鼠肝脏线粒体肿胀较轻,椭圆形,周围内质网结构尚存；AOLT组,线粒体大小不一,明显肿胀,呈类圆形。术后12h,RIPT组大鼠肝脏线粒体肿胀略加重,内嵴部分断裂,但仍有少量排列,周围内质网结构尚存；AOLT组线粒体肿胀加剧,严重者可见线粒体嵴减少、断裂或消失,周围内质网结构破坏。术后24h,RIPT组大鼠肝脏线粒体肿胀略好转,损伤未见加剧,周围内质网结构尚存；AOLT组线粒体肿胀加剧,内有空泡样变性,可见嵴断裂或消失严重。术后48小时,RIPT组大鼠肝脏细胞基本正常,见少量核碎裂相；AOLT组见较多量核碎裂、凝固及溶解,可及凋亡小体。3.5AOLT组及RIPT组血清TNF-α于术后1h均明显升高,显著高于Sham组,差异有统计学意义(P<0.05)。RIPT组血清TNF-α各时间段始终低于AOLT组,于术后1、12、24小时两者检测值差异显著,差异有统计学意义(P<0.05)。4、结论本实验采用自体原位肝移植模型,经门静脉灌肝,很好的模拟了临床上肝脏缺血-再灌注损伤的病理生理过程,避免了免疫损伤的干扰,减少了因下游脏器的缺血-再灌注损伤而受到的影响,能够相对独立的反映肝脏的缺血-再灌注损伤；板蓝根多糖灌胃预处理,能够减轻自体原位肝移植模型大鼠肝脏的缺血-再灌注损伤,其可能的机制是通过调节缺血-再灌注损伤过程中单核巨噬细胞系统的活化,尤其是肝脏Kupffer细胞的活化,抑制TNF-α等炎症介质的释放,减轻炎症细胞的浸润和对血管内皮的损伤,最终达到减轻肝脏缺血-再灌注损伤。
【Abstract】 Objective:To explore the protection effect of Radix Isatidis polysaccharide on hepatic ischemia-reperfusion injury (IRI), this empirical study in the rat autologous orthotopic liver transplantation (AOLT) model, based on the rats through the preoperative been continue intragastric administrated of Radix Isatidis polysaccharide (30mg/kg/d) of2weeks.To investigate the protection effect on the transplanted liver mediated by Radix Isatidis polysaccharide through research the changes of liver cell structure and function, the expression of correlated inflammatory factors and their interrelationship of different period, observe pathological changes and the mitochondrial structure changes of rat liver cell after the operation, detecting the expression of hepar earlier pathological changes、 biochemical indicator TNF-α and so on. Explore the possible protection mechanism of IRPS on liver of AOLT, which may provide an experiment basis for liver transplantation.Methods:2.172pure clean SD rats are includede in the study,which are divided into3groups randomly,autologous orthotopic liver transplantation model group (AOLT group n=24.);Radix Isatidis polysaccharide+autologous orthotopic liver transplantation model group (RIPT group n=24); Normal rats in control group (Sham group n=24). Two groups of liver transplantation in rats with portal vein are70%autologous transplantation model, and make technical improvements implemented since the orthotopic rat liver transplantation. Sham group are given the same treatment except the infusion.2.2Comparison of autologous orthotopic liver transplantation in rats within48h after index change.6rats were killed in1h,12h,24h,48h, after liver transplantation in each group. Collect blood samples from inferior vena cava,separate serum, detect ALT, AST and other biochemical markers with an automatic biochemical analyzer, test the expression of TNF-α by ELISA, slice of median liver lobe as a sample preparation, observe changs of liver cells’ pathological and the damage of mitochondrial and organelles in liver cells through the light microscope and transmission electron microscope. Results:3.1The criteria for successful operation: after surgery, rats immediately regained consciousness, stood up, and to maintain effective breathing, heartbeat. Portal vein perfusion, the liver warm ischemia time, generally<5s, is negligible. After the end of anhepatic phase, RIPT group of liver re-color uniform and ruddy,as the same time ALOT group then in the complex color.3.2The liver function test showed that1h after liver transplantation ALT and AST of serum both in AOLT group and RIPT group had a rapid raise.In time periods as1h12h and24h, serum ALT and AST levels of both AOLT group and RIPT group were significantly higher than that from the Sham group, there were significant differences (P all<0.05). Serum ALT and AST of the ALOT group were continuity on a high level during the time within24h after the transplantation. Serum ALT and AST of the RIPT group returned to normal as the time went on. In time periods as12h and24h, serum ALT and AST levels of the RIPT group were significantly lower than that from the AOLT group, there were significant differences (P all<0.05).3.3In each group,pathological changes of liver cells under light microscope after operation:1h after transplantation,the organizational structure of the liver changes in rats both with hepatocellular swelling, while lobular architecture is normal, the gap between the liver cells wided,hepatic sinusoid narrowed,having siltation akaryocytes and thromb,around with in the ALOT group, the organizational structure of the sham group are normal,there are no severe changes with hepatic tissue.12h after transplantation, the RIPT group has slight swelling of the liver cells, no significant changes in liver tissue; In the ALOT group, hepatocyte swelling, hepatic sinusoid narrowing and inflammatory cell infiltration is obvious.24h after transplantation, in the RIPT group,hepatocyte swelling improved. hepatic sinusoid broaden. In the ALOT group, hepatocyte swelling aggravated, hepatic sinusoid narrowing as hepatic lobules structure is not obvious, there are red blood cells and thrombus deposition, inflammatory cell infiltration appeared around;3.4Transmission electron microscopy in each group under the mitochondria of liver cells can be seen:1h after transplantation in rats from the RIPT group,the swelling of mitochondria in rats is lighter, be oval-shaped, round the remaining structure of the; in rats from the ALOT group,the mitochondria are swelling severely,be similar cyclo-shaped;after12h, in rats from the RIPT group, the swelling of mitochondria become more serious with the crest part of the fault, but still a small amount of order.the endoplasmic reticulum around are still remained, in rats from the ALOT group,the swelling of mitochondria are much more seriously,with the mitochondrial cristae decreased broken or disappeared.24h after transplantation in rats from the RIPT group,the swelling of mitochondria improved,round with normol endoplasmic reticulum;in rats from the ALOT group,vacuolar degeneration was found,severe reduction of visible ridges, broken or disappeared;48h after transplantation in rats from the RIPT group,the structure of liver cell was almost recover,a number of cells with nuclear fragmentation can be found.in rats from the ALOT group,there were much more cells with nucleolus broken solidily and dissolved,even apoptotic body can be found.3.5the level of serum TNF-α in ELISA results showed that the sham group, see a small amount of liver tissue expression of TNF-α, no significant fluctuation of the time;1h after transplantation,TNF-α expression can be seen between the sham group and the ALOT group is obvious (P<0.05),Conclusion:The autologous orthotopic liver transplantation (AOLT) model, mer-portal vein perfusion, in this empirical study, have a well simulation with the pathophysiological process of hepatic ischemia-reperfusion injury in clinically,with less interaction from immunologic injury and have less influence to the downstream organs of ischemia-reperfusion injury.can reflx independently the process of hepatic ischemia-reperfusion injury. Radix isatidis polysaccharide can protect the hepatocyte after liver transplantation by being intragastric administrated before opertion.the probable mechanism is adjusting the activation of mononuclear phagocytic system during ischemical reperfusion injury, especially the Kupffer’s cells, to refrain the dilivery of mediators of inflammation such as TNF-α, to lighten soakage of inflammatory cell and damage of blood vessel endothelium, lessen hepatic ischemia-reperfusion injury finally.