人胎肝超微结构提示肝细胞孵育红母细胞

胎肝组织电镜观察，首次发现大量红母细胞完全封入干细胞胞浆内。二者膜各自连续，其间仅留狭缝，无细胞连接，附近甚少饮液泡，紧靠红母细胞的肝细胞膜形成大量内褶，并与致密物相混形成羽毛状，提示肝细胞孵育红母细胞，此外被封入的红母细胞有提早脱核现象，且继续被孵育。     

培养人胎肝细胞的形态与功能研究

目的探讨人胎肝细胞分离与培养的简易方法．方法采用体外两步灌流法分离人胎肝细胞，选择条件培养液加以培养，并进行形态学和蛋白分泌功能的动态观察．结果用该法分离的肝细胞经台盼蓝拒染试验证实存活率在９５％以上，培养时间可达２周，并保持正常形态和良好的蛋白合成分泌功能．结论用酶灌流分离和条件培养的胎肝细胞性能良好，可用作肝细胞移植的供体．     

培养胎肝细胞上清对暴发性肝衰竭小鼠存活率的影响

目的探讨胎肝细胞悬液治疗严重肝病的机理以及使用培养胎肝细胞分泌上清（ＦＨＣＳ）替代的可能性．方法采用Ｄ＿氨基半乳糖（Ｄ＿ｇａｌｎ）／内毒素（ＬＰＳ）诱发小鼠暴发性肝衰竭（ＦＨＦ），观察ＦＨＣＳ的保护作用．结果ＦＨＣＳ能显著提高ＦＨＦ小鼠的存活率（Ｐ＜００１），降低血清转氨酶（Ｐ＜００５），减轻肝细胞坏死程度，并促进肝细胞再生．结论ＦＨＣＳ对ＦＨＦ有较好的保护与治疗作用，可望为已被迫放弃的胎肝细胞悬液输注疗法开辟新的途径     

人胎肝细胞生长因子治疗肝硬变52例

人胎肝细胞生长因子治疗肝硬变５２例邵福灵１任锡玲１李素琴２符云峰２１河北医科大学附属二院消化内科河北省石家庄市０５００００２河北医学科学院生化室ＳｕｂｊｅｃｔｈｅａｄｉｎｇｓＨｅｐａｔｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ／ｔｈｅｒａｐｅｕｔｉｃｕｓｅ...     

微载体培养人肝细胞及肝非实质细胞

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．     

大鼠肝干细胞的分离培养

目的 分离大鼠胎肝细胞,观察人肝细胞生长肽(HGP)和小鼠白血病抑制因子(mLIF)对其生长和肝干细胞集落形成的影响。方法 从大鼠胚胎(12日龄)肝中分离肝细胞,采用MTT比色法、~3H-TdR掺入法和碱性磷酸酶(AKP)检测法。结果 人HGP能剂量依赖性地促进胎肝细胞的DNA合成和增殖,500～2000u/ml的mLIF能明显促进肝干细胞集落的生长和维持未分化状态。肝干细胞培养基的组成为:5％FBS(国产或进口),10％国产NCS,0.14mM的β-ME,40ng/ml伴白蛋白,10ng/ml的人bFGF,0.5μg～1.0μg/ml的HGP,1000u/ml mLIF,1％非必需氨基酸的高糖DMEM培养基。AKP染色阳性细胞在上述培养基中传至6代仍生长良好和保持未分化状态。结论 从胎鼠肝中能分离到肝干细胞,人HGP能促进鼠胎肝细胞的生长,而mLIF对促进肝干细胞生长和维持未分化是必要的。     

肝纤维化的靶向治疗

肝纤维化是各种致病因子造成肝细胞损伤,激活库普弗细胞并使之分泌多种细胞因子,随同血小板、炎性细胞等分泌的多种细胞因子共同作用于HSC,使之转化为肌成纤维细胞；细胞因子同时激活纤维母细胞,合成并分泌大量的ECM沉积在肝Disse间隙内,抑制肝细胞生长因子及胶原酶的合成和分泌,导致肝纤维化形成,Ⅰ、Ⅲ型胶原是ECM的主要成分。[第一段]     

马肝汤对CCl_4肝损伤小鼠肝细胞修复作用的研究

目的：探讨马肝汤对肝细胞的修复作用。方法：将４０ 只小鼠随机等分为盐水对照组、药物对照组、损伤组和治疗组,损伤组和治疗组小鼠在第１、５ 天注射ＣＣｌ４ 制成肝损伤模型,药物对照组和治疗组每天皮下注射马肝汤０．１ ｍ ｌ／１６ ｇ,连续７ ｄ,各组小鼠均于第８天处死,取肝脏作电镜观察。结果：ＣＣｌ４ 肝损伤小鼠肝细胞质中含有大量的脂滴,微绒毛明显减少、肿胀,线粒体肿胀变性,血窦普遍变窄,多有灶性成堆红细胞瘀积。经马肝汤治疗后,肝细胞无明显肿胀,肝细胞质中脂滴明显减少,血窦相对增宽,无红细胞瘀积现象,肝细胞窦面微绒毛明显增多。结论：马肝汤对ＣＣｌ４ 肝损伤小鼠肝细胞具有保护、修复和再生作用     

胎肝对再生障碍性贫血患者CFU—TL的体外作用

利用T淋巴祖细胞（CFU-TL）培养系,观察了不同胎肝制剂对再障患者CFU-TL的体外作用。结果:经丝裂霉素C处理的胎肝细胞、胎肝细胞上清液及无细胞胎肝悬液均能促进再障患者的CFU-TL生长,30%～40%再障患者CFU-TL数达到正常人水平。但个别再障患者CFU-TL生长出现明显抑制效应。     

胎胸腺,脾及肝细胞悬液联合输注治疗重症肝炎42例疗效观察

近年来,国内应用人胎肝细胞悬液输注治疗重症肝炎已有不少报道,人胎胸腺、脾及肝细胞悬液联合输注治疗重症肝炎报道较少。我院近2年用以上两种混悬液输注治疗42例重症肝炎,获得较好疗效,现报道如下。     

基因修饰胎肝干细胞治疗肝纤维化

目的探讨肝细胞生长因子（HGF）基因修饰的胎肝干细胞治疗肝纤维化大鼠的效果。方法构建大鼠HGF基因和绿色荧光蛋白基因共同表达的融合载体,转染到胎肝母细胞中。将能够表达HGF的细胞移植到CCl4肝纤维化大鼠体内,将增强型绿色荧光蛋白（EGFP）修饰的胎肝干细胞作为对照组,检测大鼠肝功能和血清HGF及TGFβ1的浓度,肝组织中荧光蛋白数量和分布,肝组织内Ⅰ、Ⅲ型胶原蛋白的沉积情况。结果组织学及血清学检测显示,与对照组相比,经HGF基因修饰的胎肝干细胞能够显著改善肝纤维化指标和大鼠肝功能（P〈0.05）。结论 HGF修饰的胎肝干细胞治疗可能成为肝纤维化潜在的治疗手段。     

Enucleation of primitive erythroid cells generates a transient population of "pyrenocytes" in the mammalian fetus

Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac-derived primitive erythroblasts mature in the bloodstream and enucleate between embryonic day (E)14.5 and E16.5 of mouse gestation. While definitive erythroblasts enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm ("pyrenocytes"), it is unclear by what mechanism primitive erythroblasts enucleate. Immunohistochemical examination of fetal blood revealed primitive pyrenocytes that were confirmed by multispectral imaging flow cytometry to constitute a distinct, transient cell population. The frequency of primitive erythroblasts was higher in the liver than the bloodstream, suggesting that they enucleate in the liver, a possibility supported by their proximity to liver macrophages and the isolation of erythroblast islands containing primitive erythroblasts. Furthermore, primitive erythroblasts can reconstitute erythroblast islands in vitro by attaching to fetal liver-derived macrophages, an association mediated in part by alpha4 integrin. Late-stage primitive erythroblasts fail to enucleate in vitro unless cocultured with macrophage cells. Our studies indicate that primitive erythroblasts enucleate by nuclear extrusion to generate erythrocytes and pyrenocytes and suggest this occurs in the fetal liver in association with macrophages. Continued studies comparing primitive and definitive erythropoiesis will lead to an improved understanding of terminal erythroid maturation.     

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.     

丁型肝炎患者肝组织中Fas/FasL和肝细胞凋亡表达

目的探讨Fas/FasL及肝细胞凋亡在丁型肝炎患者肝组织中的表达及作用.方法采用免疫组化双重染色和TUNEL技术,检测79例丁肝患者肝组织HDAg,Fas/FasL表达,以及肝细胞凋亡,以54例乙型肝炎作对照.结果Fas以肝细胞浆表达为主,HDAg以核浆型表达为主,二抗原表达及分布有相关性.FasL主要表达在浸润淋巴细胞浆、肝细胞核,与HDAg表达及分布不全一致.HDAg,Fas/FasL和肝细胞凋亡在各型肝炎中的表达强度有显著性差别意义.结论HDV感染可诱导肝细胞Fas/FasL表达,增强Fas/FasL途径介导的肝细胞凋亡,这一机制在丁型肝炎发病机制中可能有一定的重要作用.     

中药虎杖对大鼠肝脏缺血性损伤保护的形态学观察

目的研究中药虎杖煎剂在治疗大鼠肝脏缺血性损伤后肝组织的病理学改变,证实该药对急性肝脏缺血性损伤有治疗作用.方法建立大鼠常温下肝门完全阻断的模型,观察肝脏缺血损伤后虎杖组和普食组在不同的时间段肝组织的病理学改变.结果通过光镜和电镜发现,术后1 d普食组与虎杖组肝细胞肿胀,结构破坏,肝窦内皮细胞孔隙加大,内皮破坏,内皮之间可见孔道.术后4 d普食组肝小叶结构仍破坏,线粒体肿胀,颗粒变性.而虎杖组未见肝细胞坏死改变,细胞膜特化部分如桥粒、毛细胆管区微绒毛有轻微破坏.术后7 d普食组肝细胞变性仍可见,线粒体轻度肿胀,基质变化,膜结构欠清楚,粗面内质网欠规则.而虎杖组肝细胞基本恢复正常形态.结论虎杖煎剂具有改善损伤肝组织的微循环,抑制白细胞、血小板与肝脏内皮细胞的粘附,达到促进肝细胞再生、修复损伤的能力.为临床上肝脏外科围手术期的应用奠定了病理学基础.     

The ultrastructural immunocytochemical demonstration of erythropoietin receptors on developing erythrocytic cells of fetal mouse liver

In spite of the severe restrictions imposed by the lack of high purity erythropoietin (Ep) and Ep specific antiserum wer have attempted to demonstrate presumptive Ep binding sites on the surface of developmental forms of erythrocytic cells of the mouse fetal liver using ultrastructural immunocytochemical methodology. Cells were exposed to exogenous Ep of differing potencies and concentrations and subsequently incubated in an Ep antiserum. The Ep-Ep-antiserum complex was then visualized with a gold-labeled IgG reagent. Limited surface labeling was noted on the developing erythrocytic cells and the evaluation of the mean surface labeling densities of the erythrocytic cell series indicated that the extent of labeling was related to the stage of maturation. Early polychromatophilic erythroblasts proved to be the most heavily labeled of the erythrocytic cells followed by the basophilic erythroblast, proerythroblast and late polychromatophilic erythroblasts respectively; normoblasts, reticulocytes and erythrocytes exhibited, at most, only minimal labeling. In addition, we have been able to demonstrate several primitive cells in the fetal liver which exhibited extensive surface labeling when compared with the developing erythrocytic cells. These cells may represent the erythroid precursors of the fetal liver.