Investigation of albumin synthesis in rat livers during the perinatal period using immunocytochemistry andin situ hybridization

The immunoreactivity of albumin (ALB) was observed in the hepatocytes of fetal rats on day 18 of gestation, and was especially observable in immature rough endoplasmic reticulum (rER) and Golgi apparatus (GA); by then, a small amount of silver grains of ALB mRNA could already be detected. Just after birth, immunoreactivity of ALB could be observed in fine granules or diffusely in all hepatocytes, and was present in rER and GA. One week after birth immunoreactivity of ALB was observed in all hepatocytes and was visible in developed rER and GA; the grains of ALB mRNA were present in all hepatocytes. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

丁酸钠体外诱导小鼠胚胎干细胞分化为肝细胞

目的:探讨分化刺激剂丁酸钠对体外培养的小鼠胚胎干细胞（ES细胞）分化为肝细胞的影响，并分析其可能机制。 方法:常规培养E14 ES细胞后，继续悬浮培养4 d以形成拟胚体，然后转移拟胚体到铺有明胶的6孔板中并添加3 mmol/L丁酸钠开始诱导分化，分化过程中用倒置相差显微镜观察细胞形态学的改变；免疫荧光染色观察肝细胞特异性标志白蛋白(ALB)、甲胎蛋白(AFP)、细胞角蛋白18（CK18）的表达；RT-PCR检测肝细胞特异性标志基因ALB、AFP、甲状腺素运载蛋白(TTR)、α1抗胰蛋白酶(AAT)的mRNA表达；流式细胞技术检测ALB阳性细胞比例；糖原PAS染色和吲哚氰绿（ICG）摄取试验检测肝细胞功能。 结果:诱导分化过程中ES细胞逐渐出现肝细胞样改变；诱导分化第7d仅检测到AFP和TTR在mRNA水平的表达，第14 d检测到ALB、AAT在mRNA水平的表达；分化14 d时免疫荧光检测显示ALB、AFP、CK18均为阳性；流式细胞技术检测ALB阳性细胞比例约为48.6%；糖原PAS染色和ICG摄取试验显示分化18d的肝细胞样细胞具有肝细胞功能。 结论:丁酸钠可以高效诱导小鼠ES细胞分化为肝细胞样细胞，并且分化细胞具有肝细胞功能。     

大鼠肝大部切除与D-氨基半乳糖肝中毒后肝细胞增殖及甲胎蛋白和白蛋白免疫细胞化学研究

大鼠肝大部切除后和D-氨基半乳糖肝中毒后第1～7天,逐日取肝组织及血液样本,观察两种类型损伤后肝再生中肝细胞的增殖分化,以及血清甲胎蛋白(AFP)与白蛋白(ALB)浓度变化及其免疫细胞化学变化。结果表明:1.肝大部切除后肝细胞分裂高峰(1.5％)出现于术后第2天,半乳糖肝中毒组的高峰在第3天,且峰值仅为前者的一半(0.8％)。2.半乳糖肝中毒后,门管区和小叶周边带出现许多小型细胞,而大部切除后的肝内无此种小型细胞。3.AFP与ALB免疫细胞化学观察表明,两种损伤后肝再生中均有AFP阳性肝细胞,于第2～3天增多,第4天后渐少。部分小型细胞呈AFP阳性。电镜下见粗面内质网、滑面内质网、高尔基复合体及核周隙等处显示AFP阳性。ALB阳性细胞第1～3天较少,于第4天起渐增多。4.血清AFP含量于第1天开始升高,第4天达高峰,此后下降,ALB含量变化恰与AFP相反,第3天下降至最低点,此后回升。     

ChAT-like immunoreactivity of olivocochlear fibres on rat outer hair cells during the postnatal development

Summary Several studies present a great deal of information about putative efferent neurotransmitters and their distribution in the adult and developing cochlea. Anatomical mapping of outer hair cell efferent fibres during ontogeny is still not available. Using quantitative electron microscopy in combination with immunocytochemistry, the distribution of ChAT-like immunoreactivity in the developing rat was investigated. Adult-like immunoreactivity in the whole cochlea is first observed in 30-day-old rats. We localized the adult-like immunoreactivity in all efferent fibres and synapses of the outer hair cells along the entire cochlear duct. An adult-like reaction in the whole cochlea could be observed on the 25th day after birth in two out of three cases. On the 20th postnatal day, no adult-like ChAT immunoreactivity was found, with the exception of one case where labelling was seen in the basal region only. The adult-like ChAT immunoreactivity on the 30th day, 2–3 weeks after the onset of hearing, is the latest maturation of all features of the organ of Corti so far investigated. Synaptogenesis of the outer hair cell efferents reaches an adult-like appearance already on the 16th day after birth.     

Electron microscopic radioautographic study on the syntheses of DNA, RNA and protein in the livers of aging mice

The syntheses of DNA, RNA and protein in the livers of aging mice was studied by electron microscopic radioautography using radioactive precursors. Labeling with³H-thymidine was observed in the nuclei of some hepatocytes at various prenatal and postnatal ages. The percentage of labeled cells decreased after birth, then slowly fell to the lowest value at 2 years. The silver grains with³H-uridine labeling were observed in both the nuclei and cytoplasm of hepatocytes at various ages. There was a peak in uridine labeling at 14 days, and then it slowly decreased until old age. The number of silver grains with³H-leucine labeling in the cytoplasm of hepatocytes was small. It increased after birth, reached the maximum at 1 month, and continued to decrease with aging.     

人胚胎肝细胞的体外培养及表型观察

目的观察体外培养的人胚胎肝细胞的特性。方法采用胰酶分步消化法体外分离人胚胎肝细胞,在培养的不同代数,收集培养上清液测定甲胎蛋白、白蛋白及5种特异性酶(ALT、AST、GGT、ALP、LDH)的分泌量,用免疫组化法测定细胞内细胞色素C的表达情况。观察诱导分化剂丁酸钠对该细胞的作用效果。结果体外分离培养的肝细胞在DMEM培养基中呈单层多边形上皮样生长,常规传代可维持约5个半月(30代),在传代过程中具有分泌白蛋白及5种特异性酶的生理功能。结论建立的人胚胎肝细胞系,保持了肝细胞的表型及分泌多种特异性酶。     

Transient expression of insulin-like growth factor I immunoreactivity in skeletal muscle cells during postnatal development in the rat

The immunohistochemical expression of insulin-like growth factor I (IGF-I; somatomedin C) was investigated in hindlimb skeletal muscle of rats during postnatal development. IGF-I immunoreactivity could be demonstrated in satellite fibres and myotubes 1, 2 and 3 days after birth, while no IGF-I immunoreactivity could be demonstrated in the more mature primary fibres. Five days after birth only scattered cells showed IGF-I immunoreactivity and 10 days after birth no specific IGF-I immunoreactivity could be demonstrated in muscle cells, i.e. the adult pattern was established. It is concluded that IGF-I immunoreactivity is expressed during a limited period of postnatal skeletal muscle maturation in rats. IGF-I is probably synthesized by IGF-I immunoreactive muscle cells and contributes to the differentiation/maturation process by autocrine and/or paracrine mechanisms.     

Differing expression of insulin-like growth factor I in the developing and in the adult rat cerebellum

Insulin-like growth factor I (IGF-I; somatomedin C) is a trophic peptide of importance for the development of several tissues and organs. In the present study we have mapped the cellular distribution and dynamic changes of IGF-I immunoreactivity in the rat cerebellum from its postnatal development to maturity. In vitro hybridization of IGF-I mRNA was used to demonstrate that the IGF-I immunoreactive material was synthesized in the cerebellum during a limited time period of cerebellar differentiation. IGF-I immunoreactivity was absent in primordial nerve cells but was present in neuroglial cells during the first two days after birth and then rapidly increased in intensity in the latter during the next few days. Proliferative nerve cells in the external granular layer did not express IGF-I immunoreactivity, while migrating and differentiating nerve cells as well as neuroglial cells showed intense labelling. Starting about 2 weeks postnatally, the IGF-I immunoreactivity declined, first in the neuroglial cells and eventually in the nerve cells. No IGF-I immunoreactivity could be demonstrated in the normal adult cerebellum. Colchicine pretreatment did, however, enable demonstration of IGF-I immunoreactivity in adult cerebellar nerve cells but not in neuroglial cells. In vitro hybridization revealed IGF-I mRNA in the developing cerebellum but only at very low levels in the adult cerebellum. It is concluded that IGF-I is likely to be a factor of importance for the development and maturation of nerve cells and neuroglial cells in the brain. The neuroglial cells in normal adult cerebellum as well as in other parts of the central nervous system do not show any IGF-I immunoreactivity, in contrast to neuroglial cells in the automatic and peripheral nervous systems.     

大鼠肝促性腺激素释放激素受体的定位研究

目的探讨大鼠肝是否表达促性腺激素释放激素(GnRH)受体,为GnRH能否参与肝代谢功能的调节提供形态学依据。方法采用兔抗GnRH的抗独特型抗体的免疫组织化学SABC法及地高辛标记探针的原位分子杂交法,对5例大鼠肝GnRH受体表达进行研究。结果大鼠肝细胞既呈GnRH受体阳性又有GnRH受体mRNA杂交信号。GnRH受体阳性物质和GnRH受体mRNA杂交信号物质均分布在大鼠肝细胞的细胞质,细胞核未检测到GnRH受体阳性物质及GnRH受体mRNA杂交信号。不同部位的肝细胞其GnRH受体免疫反应强度和GnRH受体mRNA杂交信号强度存在差别。结论大鼠肝细胞能表达GnRH受体,不同部位的肝细胞对GnRH受体表达水平存在差异。     

Occludin expression and tight junction strand formation during replicative DNA synthesis in primary cultures of rat hepatocytes

Occludin, an integral membrane protein, is a candidate for forming the functional intercellular seal of the tight junction. In the present study, we examined the changes of occludin expression and tight junction strand in the cultured hepatocytes. In L-15 medium containing EGF with 2% DMSO and 10^–7M glucagon, the great majority of the cells were quiescent. Under this condition, occludin immunoreactivity was observed at cell-to-cell contacts. Tight junction strands formed well-developed networks in freeze-fracture replicas. When the cells entered S phase of the cell cycle, occludin immunoreactivity and the number of tight junction strands decreased. By contrast, when DNA synthesis was then inhibited by readdition of DMSO and glucagon, occludin immunoreactivity and the number of tight junction strands were restored. Regardless of DNA synthesis, however, there was no significant decrease in the protein and mRNA levels of occludin. These changes of occludin immunoreactivity were correlated to the organization of actin filaments. On the other hand, no change of ZO-1 immunoreactivity was observed. These results suggested that tight junctions of cultured hepatocytes regulated in a cell cycle-dependent fashion and correlated with the organization of circumferential actin bundles.     

Sulfation and transport of basement membrane proteoglycans,as visualized by 35S-sulfate radioautography in the endodermal cells of the rat parietal yolk sac

In the hope of clarifying the biogenesis of basement membrane proteoglycans, an injection of ³⁵S-sulfate was given to concepti of 12-day gestant Sherman rats. The parietal wall of the yolk sac (including endodermal cells and the associated basement membrane known as Reichert's membrane) was removed at times varying from 7 min to 24 hr after injection and processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Between 7 min and 2 hr after ³⁵S-sulfate injection, radioactivity was observed in the endodermal cells, while from 4 to 24 hr it was mostly present in Reichert's membrane. Detailed distribution of the cellular radioactivity at 7 and 15 min showed about 20% in the rough endoplasmic reticulum (rER), 60% in the Golgi apparatus, and 8% in secretory granules. The radiactivity present in rER and Golgi apparatus decreased to low levels by 2--4 hr after injection. In secretory granules, radioactivity increased to reach a peak at 2 hr and then declined; moreover, only the granules associated with the trans Golgi face were radioactive at early time intervals, while those scattered through the cytoplasm and along the cell surface became radioactive at later times. Between 4 and 24 hr, radioactivity became negligible over all cell organelles, while it was collected in Reichert's membrane. Biochemical reports indicate that when ³⁵S-sulfate is added to organ cultures of Reichert's membrane and endodermal cells, about 90% of the incorporated which these proteoglycans acquire sulfate are likely to be those labeled at 7 min after ³⁵S-sulfate injection, that is, the Golgi apparatus and to a lesser extent the rER, whereas some labeling of the secretory granules located at the trans Golgi face is explained by rapid acquisition of sulfated proteoglycans from the Golgi apparatus. Label later appears in the secretory granules along the cell surface and, eventually, in Reichert's membrane. It is concluded that secretory granules transport newly formed proteoglycans from the Golgi apparatus to the outside for deposition into Reichert's membrane.     

原代培养肝细胞对登革病毒易感性的研究

目的 探讨登革病毒在原代肝细胞中的增殖规律及感染后肝细胞损伤特点。方法以Ⅱ型登革病毒感染原代肝细胞,采用空斑试验、生化方法和形态学方法检测病毒在细胞内的增殖及肝细胞损伤特点。结果感染后2～6d的培养上清液内均可检测到较高的病毒滴度,波动在10^5-10^6／mL。免疫荧光染色发现：感染早期细胞内有少量的病毒抗原,在核周分布,感染晚期细胞内病毒抗原量明显增多,阳性反应呈斑块状,分布在核周和胞浆内。登革病毒感染后,培养上清液中3种肝细胞分泌酶也有不同程度的变化,以乳酸脱氢酶变化最为明显,感染后1d,即呈现少许上升趋势,以后逐渐上升,7d达峰值,为20．75U／L。血清白蛋白在感染后晚期明显升高,达3．20％。谷丙转氨酶在感染3～7d升高,峰值达22．23U／L。结论原代肝细胞可支持登革病毒在其中增殖,感染后肝细胞有现明显损伤。     

皮肤成纤维细胞体外长期培养及肝向分化研究

目的 本研究通过体外长期培养皮肤成纤维细胞,试图建立此种细胞的长期培养体系,探讨其向肝细胞分化的潜能.方法 从人胎上臂取皮肤组织,分离培养成纤维细胞.采用免疫细胞化学法及流式细胞术检测细胞的CD34、CD90、CD105等细胞表型;染色体分析及软琼脂克隆形成实验鉴定细胞的生物学特性.利用肝细胞生长因子(HGF)、成纤维...     

Formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in rat liver after ischemia-reperfusion: distinct localization of the two oxidatively modified products

Ischemia-reperfusion (IR) injury is an intractable process associated not only with therapeutic recanalization of vessels, but also with partial resection or transplantation of solid organs including liver. To develop methods for predicting the degree of hepatic IR injury and further to identify injured cells, we studied the formation of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE)-modified proteins in the normothermic hepatic IR model of rats using immunohistochemistry, high-performance liquid chromatography (HPLC) determination and Western blot. The Pringle maneuver for either 15 or 30 min duration produced reversible or lethal damage, respectively. The levels of both products were significantly increased in proportion to ischemia duration 40 min after reperfusion, suggesting the involvement of hydroxyl radicals. Increased immunoreactivity of 8-OHdG was observed not only in the nuclei of hepatocytes but also in those of bile canalicular and endothelial cells. However, immunoreactivity of HNE-modified proteins was detected in the cytoplasm of hepatocytes, which was confirmed by Western blot, and in addition, in the nuclei of hepatocytes after severe injury. Thus, localization of the two oxidatively modified products was not identical. Our data suggest that these two products could be used for the assessment of hepatic IR injury in tissue, but that the biological significance of the two products might be different.     

Paucity of CFTR current but modest CFTR immunoreactivity in non-diseased human ventricle

We looked for evidence of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in non-diseased human ventricle. In rabbit and guinea pig the CFTR current is present in the highest density in subepicardial ventricular myocytes. In the present study, whole-cell patch-clamp was used to determine if a CFTR-like chloride current (I(CFTR,card)) can also be activated in human subepicardial ventricular myocytes. No evidence for I(CFTR,card) was detected in these electrophysiological studies when 10 microM forskolin was applied to 23 different cells from 4 donor hearts. Consistent with our previous results, a swelling-induced chloride current (I(Cl,swell)) could be observed after cell inflation. The enzymatic digestion of human ventricle to release single myocytes may have affected our ability to detect I(CFTR,card). Therefore, we looked for anti-CFTR immunoreactivity in slices of left ventricular free wall. A strong immunoreactivity signal was observed in guinea pig ventricle, a positive control. Background staining levels were seen in dog ventricle, a negative control tissue. Human anti-CFTR immunoreactivity was slightly above background. This low level of anti-CFTR immunoreactivity is consistent both with reports that CFTR mRNA is detectable in human ventricle and our inability to detect a significant I(CFTR,card) current density.     

不同浓度肝细胞生长因子及表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化

背景：研究证实在多种微环境下可以将骨髓间充质干细胞诱导分化为成熟肝细胞,但诱导条件及诱导分化率尚无定论,选择合适的诱导剂及诱导剂浓度尤为重要。 目的：观察肝细胞生长因子和表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化的最适浓度。 方法：不同浓度肝细胞生长因子、表皮细胞生长因子联合诱导培养原代兔骨髓间充质干细胞,观察细胞形态学变化,并检测肝细胞表面标志物甲胎蛋白、白蛋白表达及肝细胞合成功能。 结果与结论：随诱导时间延长,可观察到多极性的肝细胞样细胞。7 d时甲胎蛋白表达阳性,14 d达最大值后即开始下降(P < 0.05),此后各浓度组间表达无差别(P  > 0.05)。14 d时白蛋白表达阳性,随时间延长,阳性细胞数递增(P  < 0.05),且细胞生长因子浓度越高,阳性细胞数越高(P < 0.05)。诱导早期(9~15 d) 白蛋白上清液中白蛋白水平与诱导剂浓度成正比(P < 0.05)。18 d或21 d达高峰后下降,此时各浓度组间含量无差别(P  > 0.05)。结果显示高浓度的肝细胞生长因子及表皮细胞生长因子可提高骨髓间充质干细胞向肝细胞的分化率,诱导剂最适浓度为肝细胞生长因子60 μg/L、表皮细胞生长因子4.5 mg/L。     

Expression of neurofilament immunoreactivity in developing rat cerebellum in vitro and in vivo

The developmental expression of neurofilaments immunoreactivity was examined in frozen sections and in primary cultures of rat cerebellum by immunocytochemistry with a series of monoclonal antibodies and with a polyclonal antibody. In tissue sections immunocytochemical staining with all the antibodies used was observed in basket cells where adult-like appearance could be detected by 14 days of age and adult-level intensity was achieved by about 25 days. Granule cells remained unstained. Intense staining appeared in cerebellar white matter as early as 7 days after birth. In contrast, neurofilaments immunoreactivity was detected in cultured granule cells from 7-day-old cerebellum. Only polyclonal antibodies reacting with the highly conserved middle alpha-helical domain of the neurofilament subunits were reactive in culture. Staining could be detected in the nerve cell bodies from the first day after plating; thereafter staining intensity increased and was also distributed in neurite extensions. We conclude that unlike their counterparts in vivo cultured embryonic granule cells can express certain neurofilaments immunoreactivity.     

Immunohistochemical location of prothymosin alpha in regenerating human hepatocytes and hepatocellular carcinomas

In the present paper we analysed the presence of prothymosin alpha (ProT) in human liver. In normal liver, ProT immunostaining was found in the nuclei of bile duct cells, but not in the hepatocytes. In contrast an intense immunoreactivity was observed in regenerative hepatocytes of chronic hepatitis, cirrhosis and in hepatocellular carcinomas. In all cases the immunostaining was restricted to the nuclei, but the nucleoli were always negative. Similar results were obtained for proliferating cell nuclear antigen. These findings confirm that ProT is related to cell proliferation and provides a new immunohistochemical proliferation marker for routinely processed samples.