【作者】 朱清静；

【导师】 盛国光；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2006， 博士

【摘要】 目的： （1） 探讨丹黄方对硫代乙酰胺所致急性肝衰竭大鼠肝损伤及肝再生的作用及机制。 （2） 探讨丹黄方对部分肝切除大鼠肝再生的作用及机制。 方法： （1） 113只Wistar大鼠，雄性56只，雌性57只，体重230～250g，随机分为6组：正常对照组（8只）、急性肝衰竭组（25只）、丹黄方大剂量组（20只）、丹黄方中剂量组（20只）、丹黄方小剂量组（20只）、促肝细胞生长素组（20只）。试验第2天除正常对照组皮下注射同等剂量的生理盐水外，其余各组用TAA600mg／kg^-1体重皮下注射造模，24h后相同剂量重复注射1次。试验开始至实验结束，正常组、急性肝衰竭模型组、促肝细胞生长素组予生理盐水5ml·kg^-1灌胃，丹黄方大、中、小剂量组分别给等体积／重量比的相应药物灌胃（三组分别给予丹黄方成人等效剂量、成人等效剂量的5倍、成人等效剂量的10倍剂量进行灌胃）；除正常对照组外所有动物每日皮下注射5ml混合液（10％葡萄糖3ml、含适量生理盐水与20μmol／L KCl 2ml）以防低血糖，促肝细胞生长素组予促肝细胞生长素2mg／kg体重溶于上述防止低血糖的混合液中，每日皮下注射1次，持续3d，观察造模后48h内大鼠病死率．并于第2次注射TAA后24h后腹主动脉采血测定肝功能（ALT、AST、TB），ABC-ELISA法检测血清TNF-α水平。迅速取肝组织，用10％甲醛液中固定，石蜡切片，检测肝组织病理变化及有丝分裂指数。用链霉菌抗生物素—过氧化酶法（SP法）检测增殖细胞核抗原、肝再生信号调节蛋白α1和白细胞分化抗原14。逆转录聚合酶链反应法检测白细胞分化抗原14mRNA的表达。 （2） 雄性Wistar大鼠24只，体重230～250g，随机分为正常组（假手术对照组）、模型组（肝切除手术组）、丹黄组和pHGF组4组，每组6只。大鼠自由喂养1周后，术前12h禁食，6h禁饮。丹黄组和pHGF组于手术前3天至实验结束，每日1次分别腹腔注射生理盐水4.0ml·kg^-1体重、灌胃丹黄方10g·kg^-1体重、和灌胃生理盐水4.0ml·kg^-1体重、腹腔注射pHGF1 ml／100g，假手术对照组、肝切除手术组模型组分别给予腹腔注射0.85％生理盐水1 ml／100g，同时两组分别灌胃等体积的生理盐水。按照Higgins和Panis等介绍方法无菌条件下操作。1％戊巴比妥钠(用量40mg／kg体重^-1)皮下注入麻醉动物，固定在自制的简易手术台上，剪去上腹部鼠毛，体积分数为2％的碘酊消毒，体积分数为75％的乙醇脱碘，盖洞巾，在大鼠上腹部做一个纵形长约1cm的切口，拉出左叶和中叶，用手术线在左叶和中叶根部结扎，切除肝脏左叶和中叶（占70％），术毕皮下给予生理盐水1ml，缝合刀口，消毒。假手术组动物仅开腹翻动肝左叶和中叶，不作肝叶切除，术后自由饮食水，所有动物无1例死亡。除正常组大鼠仅进行肝叶的牵拉，其余大鼠均进行70％肝叶切除复制模型。48h后，杀死动物，迅速取肝组织，采用异硫氰酸胍一步法提取总RNA，逆转录聚合酶链反应法检测细胞癌基因fosmRNA、肝细胞生长因子mRNA、肝再生因子-1mRNA、神经生长因子诱导的抗增殖相关分泌蛋白mRNA、非受体型酪氨酸激酶mRNA和转化生长因子β1mRNA的表达。更多还原

【Abstract】 Objective（1） To study the effect and mechanism of DanHuangFang on the acute hepatic failure and liver regeneration in rats.（2） To study the effect and mechanism of DanHuangFang on the liver after partial hepatectomy in rats.Methods:（1 ） 113 Wistar rats of SPF ,weighed230 - 250 g .were randomly divided into six groups: normal control group （8 rats）, AHF group （25 rats）, DHF group of small dose （20 rats）, DHF group of middle dose （20 rats）, DHF group of large dose （20 rats）, pHGF group （20 rats）. After being fasted overnight （ over 14h）, on the second day of the experiment, all rats（except the normal group） were injected with TAA 600mgkg^-1 twice at same time for two days to induce the model of acute hepatic failure rat except the normal group injected with the 0.9 % salt solution hypodermicaily. From the first administration with TAA to the end of experiment, every rat was injected mixture solution contained 10% glucose 3ml, saline 1ml, and 20umol/L KCl·L^-1 every 8h to avoid hypoglycemia. The rats were treated with the 0.9% normal saline. DHF low, middle and high dosages, pHGF respectively every day for 3 days before TAA were injected. And observed the mortality of the rats in 48 hours, and the blood were collected to measure liver function （serum ALT, AST, TBil）,tumor necrosis factor-a （TNF-α）,after TAA were injected in the second time and the hepatic tissues were collected immediately. The pathological morphologies of hepatic tissues was observed under optics microscopy and the mitosis indexes of hepatic cells was measured. The expression of PCNA, signal regulatory protein-α （SIRP-α） and CD14 in liver cells was detected immunohistochemically , and the expression of CD14mRNA in liver was examined by reverse transcription polymerase chain reaction （RT-PCR）.（2） 24 Wistar male rats .weighed 230 250 g .were randomly divided into four groups: normal control group （shame operated group）, model group （heaptectomy group）, DHF group, pHGF group, there was six rats in every group . Before operated every rat was fasted overnight for 6h and drinking water was prohibited for 6h.From the start to the end of the experiment, every rat was treated with the 0.9 % normal saline 4.0ml·kg^-1, DHF 10g·kg^-1 dosages, pHGF 1 ml/100 g respectively every day for 3 days before operating. The rats of shame operated group and heaptectomy group were injected 85% normal saline 1ml/100g. Operation was carried according to the method of Higgins and Panis. The rats of shame operate group were turned over left and middle leaves only. There was not one rat died during the operation. And after 48 hours,更多还原