永生化人肝细胞系的建立及其生物学特性

目的 建立一种由慢病毒载体表达人端粒酶反转录酶亚单位,具有原代肝细胞的特性的永生化肝细胞系.方法 采用离体两步胶原酶灌注法无菌条件下分离肝细胞,构建慢病毒载体并包装高滴度慢病毒,肝细胞被采用表达永生化基因的慢病毒转染.通过筛选功能基因阳性表达克隆建立永生化肝细胞系,倒置相差显微镜观察形态学变化,免疫组化检测细胞角蛋白18(CK18)表达,RT-PCR及Westernblot检测功能基因及蛋白表达.结果 采用功能基因阳性表达筛选建立了永生化肝细胞系,其可连续扩增传代,具有原代肝细胞的特性并未呈现致瘤性,揭示出肝脏实质细胞的形态学特征并且表达肝细胞主要的功能基因标志.结论 通过功能基因阳性表达筛选成功建立具有原代肝细胞特性的永生化肝细胞系.     

肝衰竭血浆细胞疗法在终末期肝病中的治疗作用

以生物人工肝和肝细胞移植为代表的细胞疗法是治疗肝衰竭的一种全新策略,是继肝移植治疗急、慢性肝衰竭患者后的有效替代方法。目前细胞疗法存在的主要问题是外源性肝细胞在与肝衰竭患者血浆直接接触的交换过程中容易丧失功能,如何提高细胞材料在肝衰竭血浆中的耐受性是今后应用于临床的一个重要发展趋势。     

肝再生发生及其相关机制的研究进展

肝脏再生是指因手术、创伤、中毒、感染、坏死等致部分肝细胞功能丧失后,肝细胞重新修复的过程.对于肝再生的发生及其内在机制的认识和研究,大致可分为三个阶段:(1)最初认为可能存在一种特定的激素类物质决定肝再生的过程.     

兔慢性肝损害急性肝衰竭动物模型的建立

Objective To establish an ideal animal model of acute-on-chronic liver failure (ACLF) in New Zealand white rabbits in order to provide a large animal model for further researches.Methods Totally 75 New Zealand rabbits were randomly divided into experimental group (n =70) and control group (n = 5 ). Rabbits in the experimental group were injected with CCl4 into the abdominal cavity twice every week and the doses of CCl4 were modified according to the index of liver function and the body weight, whereas those in the control group were treated with the same volume of saline. At the 10th week,48 New Zealand rabbits with hepatic fibrosis were randomly assigned to 4 groups and injected with CCl4 as before, D-Gal at a dose of 0. 65 g/kg body weight (BW), 0. 70 g/kg BW and 0. 75 g/kg BW, respectively. By observing and comparing the general state, survival time, biochemical indexes, and the histopathology, a method of establishing a stable animal model of acute hepatic failure was found. Results As compared with those in control group, the levels of ALT, AST, GGT, HA, LN and PC-Ⅲ in the experiment group were increased significantly, while the level of ALB was decreased at the end of 10 weeks. Typical features of hepatic fibrosis and the formation of pseudo-lobules were observed at the end of 10 weeks. After treatment with D-Gal, all rabbits in group Ⅰ survived with minimal changes in liver function tests. In group Ⅱ , there was a temporary hepatic injury, but no hepatic coma. Four of the 12 rabbits died (33. 3% ). In group Ⅲ , biochemical indexes changed obviously 12 h after the administration and hepatic injury reached its peak after 48 h. Ten of 12 rabbits were died of severe hepatic failure with a survival time of ( 53. 00 ± 25. 69) h. Histology of liver section revealed massive necrosis in nodules. In group Ⅳ , hepatic injury occurred early and severely. All the rabbits died of severe hepatic failure with a survival time of (32. 70 ± 17. 46) h. Conclusion The experimental model of ACLF could be established by injected with D-Gal in New Zealand rabbits with hepatic fibrosis, induced by CCl4 intraperitoneal injection for 10 weeks.The one induced by 0. 70 g/kg of D-galactosamine was more stable and showed similar clinical pathophysiological changes in human beings. So it can be a good experimental platform for studies of ACLF.     

自体骨髓间充质干细胞移植对实验性急性肝功能衰竭的治疗作用

目的: 探讨自体骨髓间充质干细胞(me s enchymalstem cells, MSCs)移植联合内科方法对急性肝功能衰竭动物的协同治疗作用.方法: 采用D-氨基半乳糖(D-Gal)诱导建立猪急性肝功能衰竭模型. 12只实验动物随机分为4组( n = 3), 联合治疗组: 诱导24 h后, 门静脉移植5×107自体MSCs, 每24 h自体血浆100mL、甘利欣100 mg滴注; 单纯内科治疗组: 诱导后每24 h自体血浆100 mL、甘利欣100 mg滴注; 单纯自体MSCs移植组: 诱导24 h后, 经门静脉移植5×107自体MSCs; 对照组: 除普通观察护理外, 不予以任何治疗. 以对照组存活时间为整个实验观察窗口, 观察家猪的肝脏功能及病理变化.结果: 对照组全部死亡后(存活44±3.5 h), 其他3组所有实验动物人为促死. 肝脏功能指标在联合治疗组、单纯内科治疗组及单纯MSCs移植组明显优于对照组( P<0.05), 联合治疗组比较单纯内科治疗组及单纯自体MSCs移植组有明显改善( P<0.05). 肝脏病理HE染色提示联合治疗组、单纯内科治疗组病理改变轻于其他组, 联合治疗组病理改变轻于内科治疗组, 单纯MSCs移植组与对照组比较无明显差异. 免疫组织化学显示联合治疗组肝脏细胞增殖率(34%)明显优于其他组, 单纯内科治疗组、单纯MSCs移植组及对照组无明显差异.结论: 单纯骨髓MSCs移植或者内科治疗急性肝功能衰竭有一定效果, 骨髓MSCs移植联合内科治疗具有一定协同作用, 可明显改善D-Gal诱导的急性肝衰竭家猪的肝脏指标, 促进肝组织及功能恢复.     

猪骨髓间充质干细胞对共培养肝细胞的作用及其机制

目的 探索细胞外基质(ECM)在猪肝细胞与骨髓间充质干细胞(MSCs)体外共培养中的表达与分布规律. 方法自中华实验猪髂前上棘抽取骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养,观察肝细胞形态和功能的变化情况.用免疫细胞化学染色法观察ECM的表达和分布情况.RNA干扰MSCs后继续观察共培养肝细胞的功能变化情况.多组间比较采用单因素方差分析,两两比较采用LSD法.结果 第3代MSCs纯度>90%;肝细胞活率>95%,纯度>99%.共培养组肝细胞迅速黏附于MSCs表面,呈肝细胞岛样分布.共培养组肝细胞白蛋白分泌水平和尿素合成能力自共培养第1天起均明显高于单纯肝细胞组(P值均<0.01),并在第2天达到高峰,分别为(457.71士22.62)ng和(69.05±2.12)μg.免疫细胞化学检测结果显示共培养组MSCs表达多种ECMl RNA干扰实验进一步证实ECM的存在与共培养中肝细胞的白蛋白分泌和尿素合成功能有关.结论 骨髓MSCs通过分泌多种ECM改善共培养肝细胞的形态与功能.     

训练免疫在相关疾病中的研究进展

训练免疫(trained immunity,TI)是不依赖T、B细胞的,区别于传统固有免疫特征的一种记忆性固有免疫应答,在机体初次感染后对相同或不同病原体再次感染表现高反应性.训练免疫作为一种新兴的观点,具体机制涉及固有免疫、代谢及表观遗传之间的交互作用.基于此,本文总结了脂多糖(lipopolysaccharide,...     

肝再生发生及其相关机制的研究进展

肝脏再生是指因手术、创伤、中毒、感染、坏死等致部分肝细胞功能丧失后,肝细胞重新修复的过程.对于肝再生的发生及其内在机制的认识和研究,大致可分为三个阶段:(1)最初认为可能存在一种特定的激素类物质决定肝再生的过程.     

输尿管非结石性梗阻病因及诊断分析

目的提高对输尿管非结石性梗阻的诊断水平.方法回顾性分析近10年来收治的输尿管非结石性梗阻患者的病因及诊断方法.结果损伤性输尿管狭窄72例,输尿管癌22例,输尿管囊肿18例,输尿管结核13例,先天性肾盂输尿管交界处狭窄8例,非特异性输尿管炎7例,输尿管息肉6例.结论输尿管癌为中老年输尿管非结石性梗阻患者的首要病因,损伤性输尿管狭窄为青年患者的首要病因.通过病史和恰当的检测手段,可以明确病因.