小鼠胎肝基质细胞体外扩增骨髓来源的LTC-IC

基质细胞是胎肝造血微环境的主要成分,参与造血干/祖细胞的自我更新、增殖分化的调控。为了研究小鼠胎肝基质细胞在造血微环境中的功能,采用转染SV40大T抗原基因的方法建立了小鼠胚胎期第12.5天（Embryonic-day 12.5, E12.5d）胎肝基质细胞系A4、B3,并进一步鉴定基质细胞系的一般细胞生物学特性和造血支持功能。结果：A4、B3为细胞形态、生长行为以及表面分子表达不同细胞系,二者均可维持骨髓源长期培养启动细胞（Longterm cultureinitiating cell,LTC-IC）至少4周并且有不同程度的扩增LTC-IC能力,其中B3扩增LTC-IC的能力是A4的83倍。外源性细胞因子组合SCF+IL-3+IL6+Epo在本实验体系中不影响LTC-IC数量的维持和扩增。暗示E12.5d胎肝造血微环境中基质细胞的功能是不同的,其机制有待进一步研究。     

人胎肝干细胞的分离培养、鉴定及mRNA转录分析

目的：探讨体外大量扩增培养人胎肝干细胞的方法,研究其形态、特性及mRNA转录情况。方法：采用两步灌流法结合链霉蛋白酶消化及Percoll密度梯度离心法分离12～20周胎龄的胎肝细胞,采用免疫细胞化学染色及RT-PCR方法对分离培养的细胞进行鉴定分析。结果：刚分离的细胞活力在80%以上,原代培养3d开始出现小细胞团,2周后即形成肉眼可见的细胞集落,细胞体积小,核质比大;原代、传代培养的胎肝细胞甲胎蛋白（AFP）、细胞角蛋白19（CK19）、卵圆标记蛋白OV-6、细胞分化抗原34免疫染色阳性;RT-PCR分析证明胎肝干细胞中AFP、白蛋白、CK19、CK18和八聚体结合蛋白（OCT）-4mRNA的表达。结论：分离了胎肝干细胞,具有肝细胞、胆管细胞及干细胞表面标志及相应的基因表达,为进一步的基础研究奠定了基础。     

大鼠肝癌(BERH-2)细胞甲胎蛋白信使核糖核酸的分离与提纯

采用双抗体免疫沉淀合成AFP的聚核糖体并合Poly(U)-Sepharose亲和层析的方法分离和提纯了大鼠肝癌BERH-2细胞的AFPmRNA。这种纯的AFPmRNA在2％聚丙烯酰胺—0.5％琼脂糖凝胶中以单一的22S带移动。用双抗体免疫沉淀的方法,鉴定麦胚抽提液中由AFPmRNA直接合成的蛋白。在纯甲胎抗体所沉淀的多肽中,有95％的放射性在凝胶电泳中与载体AFP一起移动。在最佳条件下,AFPmRNA在麦胚系统中翻译产物比AFPpRNA高54倍。用AMV反转录酶合成互补于AFP-mRNA的DNA(AFP~3H-cDNA),用分析AFP-mRNA.AFP~3H-cDNA杂化物的热变性来测定AFPcDNA的真实性。高达89℃的Tm值,表明所反转录的cDNA是AFP-mRNA真实的拷贝。     

肝纤维化的细胞和分子机制

肝脏纤维化(hepatic fibrosis)是多种慢性肝病的共同病理基础,是进一步向肝硬化发展的中心环节。肝脏内一些免疫细胞如枯否细胞(Kupffer cell,KC)、树突状细胞(dendritic cell,DC)、T淋巴细胞、NK细胞(nature killer cell,NK cell)、B细胞等在多种致病因素刺激下激活,释放多种细胞因子和趋化因子,引起一系列病理变化,共同参与肝纤维化的发生和发展过程。本文主要从肝脏内各类免疫细胞以及分泌的细胞因子方面,对肝纤维化形成机制的最新研究进展进行综述。     

单克隆来源的小鼠胎肝HPP-CFC肝上皮分化潜能的研究

为研究胎肝中造血和肝上皮发育的关系,建立了小鼠胎肝高增殖潜能集落形成细胞（HPP-CFC）培养体系,并进行了单克隆培养以及诱导分化实验.在造血和肝诱导因子的共同作用下,对单克隆来源的HPP集落细胞向造血和肝上皮细胞进行诱导分化,采用透射电镜(TEM)、巢式RT-PCR、细胞免疫荧光检测,从细胞形态、超微结构、上皮细胞分化标志等方面对分化后的细胞进行检测.检测结果显示诱导后的部分细胞具有肝细胞特异性的超微结构并不同程度的表达白蛋白（ALB）、甲胎蛋白（AFP）、细胞角蛋白（CK8,CK18）等肝上皮分化标志,同时还表达间质标志α-SMA和血管内皮细胞标志Flk-1.免疫磁珠分选表明：胎肝来源的HPP-CFC主要来自于CD45⁺细胞,CD45^-细胞不具有形成造血克隆的能力.在肝上皮细胞分化潜能上,流式分选获得的CD49f⁺/Sca-1⁺细胞与未分选细胞无明显差异.该模型的克隆源性通过细胞混合实验进行证明.研究结果表明,改进的胎肝来源的HPP-CFC可能代表了一个新的造血向肝上皮细胞分化的单克隆模型,为研究胎肝中造血和非造血细胞的发育关系提供了一个新的切入点.     

Zn2+对中华绒螯蟹溞状幼体肝胰腺超微结构的影响

研究了不同Zn2+浓度对中华绒螯蟹(Eriocheir sinensis)()状幼体肝胰腺细胞超微结构的影响.当Zn2+浓度超过200 μg/L时,与对照组相比,肝胰腺结构受到了显著的影响.成熟和正在形成的B细胞的空泡中有很多含有金属的电子致密颗粒(EDG,可能为金属蛋白复合体),随着B细胞的成熟和B细胞从肝胰腺管壁上脱落,这些EDG也被释放到管腔中,因此肝胰腺的管腔中常有许多此类颗粒存在.B细胞空泡或管腔中的EDG与环境中Zn 2+浓度高低呈正相关.E细胞质中也出现了很多空泡,与正常E细胞的结构明显不同.R细胞的细胞质常解体形成大的空泡,被破坏的程度较为严重.在Zn2+浓度>1 000 μg /L时,肝胰腺遭到严重破坏,细胞结构几乎不存在.研究结果表明,肝胰腺的B细胞在Zn 2+的解毒方面起重要作用,但当Zn2+浓度超过了肝胰腺B细胞的解毒能力时,就会引起肝胰腺细胞(如R和E细胞)结构异常,甚至破坏整个肝胰腺细胞结构,从而影响幼体的生长,甚至造成死亡.     

胎肝滤液诱导大鼠胎盘间充质干细胞向肝细胞的分化

目的探讨PDMSCs向肝细胞增殖和分化的体外培养条件及方法。方法孕20 d的大鼠无菌条件下取胎盘,经胶原酶消化、密度离心、贴壁筛选法分离培养胎盘源间充质干细胞,并对其表面抗原进行鉴定。在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导PDMSCs向肝细胞定向分化,以免疫细胞化学检测干细胞标志物;PAS检测糖原表达。结果在体外培养条件下,PDMSCs贴壁生长为成纤维样细胞,CD44表面标志物检测阳性;PDMSCs经胎肝滤液诱导14d时细胞呈现圆形、卵圆形的特征性改变,AFP、CK19表达阳性。结论胎肝滤液能够诱导PDMSCs定向分化为肝细胞样细胞。     

胎儿脾内B淋巴细胞发育的研究

本文利用一系列抗体和免疫组织化学,在冰冻切片上对不同胎龄(9～38周)的脾,比较观察了淋巴组织形成过程中,B细胞膜抗原的变化。结果发现胎儿脾内T、B淋巴细胞集聚是沿血管分布的,开始为集落样,随着淋巴细胞不断增加,则分别发育为脾小结和动脉周围淋巴鞘。B细胞由集落发展为脾小结时,膜抗原出现一系列变化,如Leu14和BA-1由弱阳性到阳性,OKB-2和Tac由阴性转变为阳性。其他的各种SIg,HLA-DR也有相应改变,这些膜抗原表达的变化,提示B细胞向成熟方面发育,但未发育为浆细胞,脾小结内也未见有生长中心。T细胞数量由少到多,但染色强度没有改变,这是因为T细胞不在脾内发育,而且进入外周淋巴器官的T细胞,功能上是成熟的。     

胎鼠肝脏发育过程中肝窦的形成与成熟

为了研究胎鼠肝脏发育过程中肝窦的形成与成熟,用窦内皮细胞表达的一种特异性抗原,SE-S,作为窦内皮细胞表型成熟的标志,通过免疫组织化学和免疫电镜来观察肝脏发育的不同时期该抗原的表达以及与先存大血管的关系。实验证明,在胎龄15天时,即可观察到散在的肝窦样结构呈现阳性反应,而表达第八因子相关抗原的大血管为阴性。随着肝脏的发育,表达SE-S抗原的肝窦在数量和长度上亦随之增加,并且与肝细胞直接或密切接触。结果提示,肝窦的形成与成熟似乎是随机发生的,可能始于胚胎发育的第15天或更早,与先存大血管无直接相关性;肝细胞可能调节窦内皮细胞的成熟(SE-S抗原的表达)。     

大鼠3′-甲基-4,4′-二甲基氨基偶氮苯(3′-MeDAB)诱癌过程甲胎蛋白的动态变化和免疫酶标记定位观察

本工作采用放射火箭电泳自显术和免疫酶标记定位技术,对大鼠3'-MeDAB诱癌过程血清和肝组织甲胎蛋白(AFP)的动态变化和定位情况进行了观察。发现(1)肝硬化假小叶内的少数肝细胞、嗜碱性间变再生结节细胞、少数“幸存肝细胞”及少数“过渡性细胞”,具有合成AFP的能力。未见肝内其他类型细胞合成AFP。合成AFP的细胞大多具有胞浆嗜碱性、生长活跃和形态上去分化的特点。(2)未见胆管癌细胞合成AFP。肝细胞癌分化好的癌细胞绝大多数也未见合成AFP,分化差的癌细胞合成AFP的能力,基本上与其生长活跃程度呈正相关,与分化程度呈负相关,而与癌细胞是否处于核分裂阶段关系不大。(3)肝癌组织AFP酶标的强度和范围与血清AFP水平之间基本上有平行关系。此外,本文还就什么细胞合成AFP、血清AFP“马鞍型”变化的成因及病理组织学基础等进行了讨论,并根据实验结果,对大鼠3'-MeDAB肝癌的组织发生提出了初步的设想。     

Ontogeny of the antibody-forming cell line in mice. IV. Appearance of cells bearing Fc receptors, complement receptors, and surface immunoglobulin.

The ontogeny of Ig, FcR, and CR-bearing cells in liver and spleen has been followed by using rosetting procedures. These studies demonstrated a sequential appearance of surface receptors during development. Two types of Ig+ cells could be distinguished according to their rosette morphology and adherence to carbonyl iron: 1) an adherent cell which bound few erythrocytes was found predominantly in fetal liver from 13 days gestation and 2) a nonadherent cell which bound larger numbers of erythrocytes appeared in small numbers in fetal liver from day-16 gestation but represented the major Ig+ cell type after birth. Changes in the proportions of receptor-bearing populations occurred at two particular periods during ontogeny. The first was at birth, where an increase in the proportion of FcR+ cells occurred and the proportion of type 2 Ig+ cells rose rapidly. This probably represented the first appearance of FcR+ B lymphocytes even though cells bearing FcR were detected in fetal liver of all ages (days 12 to 18). The second period was around 10 days after birth when the proportion of Ig+ cells again increased concomitant with the appearance of CR+ nonadherent cells.     

人胎脾交错突细胞对T,B淋巴细胞发育影响的免疫组织化学研究

用免疫酶单重和双重染色研究人胎儿脾连续切片中交错突细胞（ＩＤＣ）与Ｔ,Ｂ淋巴细胞的定位关系及ＨＬＡ－ＤＲ表达。结果表明,Ｓ－１００阳性树突状细胞为ＩＤＣ,多数表达ＨＬＡ－ＤＲ。９－１２周的胎脾中就可见到散在分布的ＩＤＣ。１３－１６周胎脾中ＩＤＣ开始定位于白髓的Ｔ细胞集落内和周缘,及Ｂ细胞集落的周边。在上述区域ＩＤＣ常与Ｔ细胞形成ＩＤＣ－Ｔ细胞聚合体。在脾的发育过程中,ＩＤＣ不仅与Ｔ,Ｂ淋巴细胞在分布上关系密切,而且可与这两类细胞形成突起－胞体、胞体－胞体的连接。提示,胎儿脾中ＩＤＣ与Ｔ,Ｂ细胞的迁移,定位及功能成熟过程有密切联系。     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Interaction of trans-acting factors with the proximal promoter of the mouse alpha-fetoprotein gene.

1. The alpha-Fetoprotein (AFP) gene is expressed during fetal life, but not in adult cells. Also, the AFP gene is expressed in most hepatomas. 2. Using gel retardation (band-shift) assays under very stringent conditions we have compared the binding of trans-acting factors to the proximal enhancer (-202, +34) region of the AFP gene. 3. We have detected the presence of two retarded bands in experiments performed with adult rat hepatocytes and the Fa32 cell line (which does not produce AFP) but only one band is observed with the HepG2 cell line (which produces AFP) and fetal liver. 4. We relate the two retarded bands to a glucocorticoid response element and, tentatively, to the C/EBP trans-acting fractor.     

Cellular immunolocalization of alpha-fetoprotein in rat liver

Increased synthesis of alpha-fetoprotein (AFP) was induced in rat liver by the administration of 3'-methyl-4-dimethyl-aminoazobenzene. The indirect immunoperoxidase technique was used to detect AFP. Cellular localization of AFP was studied using a number of different fixation procedures. Serial sections stained with immunoglobulin served to determine the extent of diffusion of serum proteins into liver cells during fixation. Background staining was minimized when Lillie's neutral buffered formalin plus acetic acid was used as the fixative. After 3'-methyl-4-dimethylaminoazobenzene ingestion, bile duct cell proliferation occurred. The serum AFP was positive in all rats after 17 days on the diet. In rats with AFP-positive sera the immunohistochemical reaction in mature hepatocytes was positive while bile duct cells and small hepatocytes were negative for AFP.     

Kinetics of albumin- and alpha-fetoprotein-production during rat liver development

Synthesis of most of the plasma proteins is one of the main functions of the hepatocytes. Albumin synthesis is quantitatively the most abundant. In the present study we investigated albumin- and alpha-fetoprotein-gene-expression, and the function of the secretory apparatus during rat liver development. To this purpose we used the method of radioactive biosynthetic labeling of newly synthesized albumin and alpha-fetoprotein (AFP) to monitor the secretory capacity of endodermal cells derived from ventral foregut region (embryonic day 10, E10), and of embryonic and fetal hepatoblasts. Synthesis and secretion of albumin and AFP were already detected in the low numbered ventral foregut endodermal cells; fibrinogen synthesis was detectable in the E12 hepatoblasts, which were in higher number. The whole secretory machinery was functional from the earliest stages of liver development, and the speed of secretion was comparable with that of the adult hepatocytes. There was almost 4-fold increase of hepatoblasts cell volume in fetal stage compared with embryonic stage. The model used suggests that the hepatocyte secretory apparatus is already functional before the emergence of the liver bud. This is the first comparative report to analyze the hepatocyte secretory function, cell proliferation and cell volume during liver development.     

Hepatic lineages isolated from developing rat liver show different ways of maturation

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.