Characterization of the beta-adrenergic receptor in isolated human fetal lung type II cells.

Functioning of the beta-adrenergic response system is important for successful transition of the neonate from fetal life to breathing air. We characterized the beta-adrenergic receptors on human fetal lung type II cells, the cell type responsible for many pulmonary responses sensitive to beta-adrenergic stimulation. Type II cells were isolated from human fetal lung explants, and membrane particulates prepared from these cells were used for radioligand binding studies. 125I-iodocyanopindolol, a specific beta-adrenergic antagonist, bound to a single class of saturable, high-affinity binding sites on type II cell membranes with a receptor concentration of 78 +/- 9 fmol receptor/mg membrane protein, a kd of 79 +/- 18 nM, and 958 +/- 120 receptors per cell. Binding was stereoselective with l-propranolol binding with higher affinity than the inactive d-isomer. The binding site had the characteristics of a beta 2-adrenergic receptor. The order of potency of beta-adrenergic agonists was isoproterenol greater than epinephrine much greater than norepinephrine. The beta 2-selective antagonist ICI 118,551 competed for a single class of high-affinity sites. Agonist binding affinity was reduced in the presence of guanyl nucleotides, consistent with receptors coupled to guanine nucleotide binding proteins. beta-Adrenergic agonists also stimulated adenylyl cyclase in these membrane preparations. 125I-iodocyanopindolol binding to membranes prepared from human fetal lung fibroblasts indicated fewer receptors (404 +/- 68) than were present on type II cells. Work by others has suggested a difference in lung function and lung beta-adrenergic receptor concentration between males and females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surfactant content and type II cell development in fetal guinea pig lungs during prenatal starvation

Prenatal caloric restriction in guinea pigs causes intrauterine growth retardation and reduced neonatal viability and surfactant phospholipid (PL). We report here fetal surfactant levels in this model, and correlate total lung PL with ultrastructural maturation of surfactant type II cells and lamellar bodies (LB). Pregnant guinea pigs were fed ad libitum throughout their 68-d gestation (control), or fed 50% rations from d 45 until term (starved). Fetal lungs were examined at d 55, 60, and 65 for PL content and composition, including disaturated phosphatidylcholine (DPC), and compared with neonates for both groups. Lung lobes were analyzed ultrastructurally in d 65 fetuses for the numerical, volume, and surface densities of type II cells and the volume densities of LB. Prenatal starvation caused significant intrauterine growth retardation at all ages; body and dry lung weights were reduced on d 65 by 26 and 23%, respectively. By d 55 and thereafter, starvation decreased total lung PL by 43-45% but did not alter PL composition. On d 65, the total lung volumes and relative numbers, surface densities, and volumes of type II cells in tissue and the relative volumes of LB within type II cells did not differ by caloric regimen. Thus, starved and control fetuses had similar total volumes of LB per lung (13-15 microL), although starved animals had significantly less lung DPC. Although the total volume of LB per lung correlated well with total lung DPC from d 55 through birth in controls, starvation led to a significant departure from this relationship.(ABSTRACT TRUNCATED AT 250 WORDS)     

核心蛋白聚糖对转化生长因子β1诱导人近端肾小管上皮细胞转分化的影响

目的探讨核心蛋白聚糖对转化生长因子β1（TGF-β1）诱导的人。肾小管上皮细胞（HK-2）转分化的影响。方法将体外培养的HK-2细胞分为6组：阴性对照组（A组）;10ng／ml TGF-β1组（B组）;10ng／ml核心蛋白聚糖组（C组）;100ng／ml核心蛋白聚糖组（D组）;10ng／ml TGF-β1＋10ng／ml核心蛋白聚糖组（E组）;10ng／ml TGF-β1＋100ng／ml核心蛋白聚糖组（F组）。倒置显微镜下观察加入刺激因子48h后,各组细胞形态学变化;应用逆转录-聚合酶链反应（RT-PCR）检测核心蛋白聚糖对TGF-β1诱导的HK-2细胞内α平滑肌肌动蛋白（α-SMA）、波形蛋白、角蛋白mRNA表达的影响。结果B组与A组相比较,细胞形态发生明显变化,大部分细胞拉长呈梭形,似成纤维细胞;E组和F组比B组梭形样细胞明显减少,尤其是F组梭形样细胞减少更明显;C组和D组与A组细胞形态无明显区别,为椭圆形;B组与A组比较,波形蛋白、α-SMAmRNA表达明显提高,角蛋白mRNA表达减少。B组平均值分别为（0.828±0.297）、（0.332±0.025）、（0.075±0.040）,A组平均值分别为（0.033±0.060）、（0.003±0.000）、（0.348±0.012）,差异有统计学意义（P〈0.05）;E组和F组与B组相比,波形蛋白、α-SMA的表达有所下降,角蛋白的表达呈上升趋势（P〈0.05）,平均值分别为E组（0.234±0.313）、（0.214±0.196）、（0.098±0.056）,F组（0.155±0.053）、（0.122±0.130）、（0.215±0.122）。C组和D组与A组比较差异无统计学意义（P〉0.05）。结论TGF-β1能诱导正常人肾小管上皮细胞发生转分化;核心蛋白聚糖对TGF-β1诱导的HK-2细胞转分化具有抑制作用,且呈剂量依赖性。     

In vitro effect of multiplication stimulating activity (MSA) on human fetal and postnatal cartilage

Although insulin-like growth factors may have a physiological role in fetal growth, little is known of their biological action on human fetal tissues. In the present study, the action of multiplication stimulating activity (MSA) on human fetal cartilage in vitro, has been examined and compared with its effect on postnatal cartilage. Addition of MSA (10-100 ng/ml) resulted in a dose dependent increase in [3H]thymidine incorporation into fetal cartilage aged between 15 and 18 weeks of gestation. The mean response with 100 ng/ml was 143 +/- 18% (n = 10) of basal levels. The increase in [35S]sulphate incorporation was variable, the mean (131 +/- 36%, n = 5) being not significantly greater than in controls. The increase in [3H]thymidine incorporation on addition of MSA was not seen in fetal cartilage of earlier (13/14 wk) or later (19 wk) gestational age. MSA-III (a highly purified component of MSA) at 100 ng/ml increased [3H]thymidine and [35S]sulphate incorporation into cartilage from a fetus of 17 weeks to 165% and 150%, respectively, but had no effect on the incorporation of either isotope into cartilage from a fetus of 19 weeks gestation. In contrast to the mitogenic effects of MSA on fetal cartilage, the same preparation had no effect on either [3H]thymidine or [35S]sulphate incorporation into postnatal cartilage. These results may reflect developmental changes in cartilage response to insulin-like growth factors similar to those reported in human brain.     

Angiotensin II and basic fibroblast growth factor mitogenic pathways in human fetal mesangial cells

Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF/FGF-2) play relevant roles in renal development. Since the signaling pathways modulating the mitogenic effects of Ang II and bFGF in human fetal mesangial cells (HFMc) are not clearly defined, we carried out experiments to determine whether they would exert their mitogenic effects by modulating the activity of the mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase-2 (ERK-2)] and cAMP signaling pathways. In confluent HFMc, bFGF (20 ng/mL) induced a significant 4-fold increase in ERK-2 activity and [3H]-thymidine incorporation (6-fold). In contrast, under similar tissue culture conditions, Ang II (10(-6) M) induced a more modest increase in ERK-2 activity (2-fold) and [3H]-thymidine incorporation (35 +/- 4%). The mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD098059 (25 microM) almost completely abolished the bFGF-induced proliferation in HFMc but did not significantly affect Ang II proliferative effects. In the presence of the cAMP elevating agent isoproterenol, Ang II and bFGF induced opposite changes in cAMP accumulation and cell growth. Isoproterenol inhibited the basal and bFGF-induced proliferation of HFMc through a MEK-1/2-independent pathway that included the accumulation of cAMP. In contrast, isoproterenol increased Ang II mitogenic effects in correlation with a reduction in cAMP accumulation. We conclude that Ang II and bFGF modulate the proliferation of HFMc through the stimulation of different MEK-1/2-dependent and independent signaling pathways. Activation of MEK-1/2 is required but not sufficient for mitogenesis in HFMc. The accumulation of cAMP in HFMc counteracts the mitogenic effects of bFGF by a MEK-1/2-independent pathway.     

黄芩苷体外诱导脐血间充质干细胞向神经元样细胞分化的实验研究

目的探讨中药黄芩苷体外诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其可能的机制。方法采集健康孕妇足月顺产儿的脐带血,共5人份,以肝素抗凝,采用明胶沉降加密度梯度离心两步法分离脐血单个核细胞,加入含黄芩苷50μmol/L的液体培养体系中进行扩增培养。取黄芩苷体外扩增2周的人脐血MSCs,采用黄芩苷诱导24h后,继续维持诱导6d,诱导30min后开始在倒置显微镜下动态观察脐血MSCs生长情况及诱导前后形态学变化。免疫细胞化学染色评价神经细胞特异性烯醇化酶(NSE),微管相关蛋白2(MAP-2)阳性细胞的表达。诱导液(DMEM培养基,200~400μmol/L黄芩苷),37℃,5%CO2诱导24h。维持诱导液(DMEM培养基,200~400μmol/L黄芩苷,B27)继续维持诱导1周。实验共分4组,分别为诱导组;对照1组(诱导液和维持液均不含黄芩苷)、对照2组(诱导液和维持液含3mmol/L的β-巯基乙醇,不含黄芩苷)、对照3组(诱导液和维持液含20g/L二甲基亚砜和20mmol/L丁化羟基苯甲醚,不含黄芩苷)、对照4组(诱导液和维持液均含有上述浓度的黄芩苷、β-巯基乙醇、二甲基亚砜和丁化羟基苯甲醚),各组分别在诱导6h、24h、7d留取标本,制作细胞爬片,细胞固定后,免疫细胞化学染色评价NSE和MAP-2阳性细胞的表达率;Hoechest33258染色,评价细胞存活率。结果诱导组诱导6h后,细胞微丝收缩,原来梭形的脐血MSCs胞体已发生收缩,细胞边缘变得不规整,出现了细的突起。7d后多数细胞成锥形,交织成网,形成较典型的神经元细胞样形态结构,免疫细胞化学染色显示黄芩苷诱导组NSE、MAP-2阳性细胞表达率以及细胞存活率分别为(76.3±9.2)%、(78.5±5.5)%、(85.3±4.8)%,显著高于对照1、2、3组(P<0.01),分别为(4.6±0.6)%、(0.7±0.6)%、(46.7±9.2)%;(63.3±6.8)%、(40.9±5.1)%、(66.5±5.2)%和(71.6±4.7)%、(42.3±4.5)%、(72.8±7.6)%。此外,各组胶质纤维酸性蛋白(GFAP)的表达率均低于1%。结论低浓度的黄芩苷体外扩增2周后的MSCs中已有少量表达NSE的阳性细胞出现,这在一定程度上黄芩苷起到了预诱导分化作用;黄芩苷能够体外诱导脐血MSCs分化为神经元样细胞,诱导过程中黄芩苷的诱导作用温和、稳定而持久;诱导出的神经元样细胞成活时间长,其诱导机制可能与黄芩苷的抗氧化、调控细胞NF-κB的活性从而刺激多种细胞因子的表达和生成有关。     

In vitro hepatic differentiation of human umbilical cord blood and bone marrow cells

The purpose of the present study was to investigate whether human umbilical cord blood (UCB) as well as bone marrow (BM) can generate hepatocyte lineage cells in a simple culture condition. Mononuclear cells (MNCs) separated from UCB and BM were cultured in the presence of fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), and hepatocyte growth factor (HGF). The cultured cells were analyzed for morphology and for the expression of mRNAs and/or proteins of hepatocyte lineage markers. Both the UCB and BM MNCs grown in the given culture condition yielded large, round cells that were adherent to the culture dishes. RT-PCR analysis showed that mRNAs of albumin (ALB), cytokeratin (CK)-18, and alpha-fetoprotein were expressed from day 7 in both the UCB- and BM-derived cells. Immunofluorescent staining showed that the large, round cells expressed not only ALB and CK-19 but also proliferating cell nuclear antigen, implying the proliferative potential of hepatocyte lineage cells. Therefore, UCB as well as BM can give rise to hepatocyte lineage cells in the simple culture condition with HGF, SCF, FGF-1, and FGF-2. These cells may be one of the potential candidates of cell sources for the cytotherapy of hepatic disease, although it remains to be determined if the hepatocyte lineage cells are derived from plastic hematopoietic stem cells or from liver stem cells that reside in UCB or BM.     

DNA microarray reveals novel genes induced by mechanical forces in fetal lung type II epithelial cells

Mechanical forces are essential for normal fetal lung development. However, the cellular and molecular mechanisms regulating this process are still poorly defined. In this study, we used oligonucleotide microarrays to investigate gene expression in cultured embryonic d 19 rat fetal lung type II epithelial cells exposed to a level of mechanical strain similar to the developing lung. Significance Analysis of Microarrays (SAM) identified 92 genes differentially expressed by strain. Interestingly, several members of the solute carrier family of amino acid transporter (Slc7a1, Slc7a3, Slc6a9, and tumor-associated protein 1) genes involved in amino acid synthesis (Phgdh, Psat1, Psph, Cars, and Asns), as well as the amiloride-sensitive epithelial sodium channel gene (Scnn1a) were up-regulated by the application of force. These results were confirmed by quantitative real-time PCR (qRT-PCR). Thus, this study identifies genes induced by strain that may be important for amino acid signaling pathways and protein synthesis in fetal type II cells. In addition, these data suggest that mechanical forces may contribute to facilitate lung fluid reabsorption in preparation for birth. Taken together, the present investigation provides further insights into how mechanical forces may modulate fetal lung development.     

Latency in vitro of varicella-zoster virus in cells derived from human fetal dorsal root ganglia.

A potential in vitro model of varicella-zoster virus (VZV) latency was developed. Dissociated human dorsal root ganglion cultures were infected with VZV and maintained for 1 wk in the presence of bromovinyl arabinosyl uracil, a potent inhibitor of VZV. Seven to 21 d after removing the inhibitor (> or = 14 d after infection), the cells were trypsinized, passed to monolayers of human embryonic lung fibroblasts, and observed for VZV reactivation as indicated by typical cytopathic effects and the appearance of VZV antigens. VZV reactivated from 56% of the cultures containing both neurons and satellite cells but not from cultures specifically enriched for either neurons, satellite cells, or ganglion-derived fibroblasts. The failure to isolate VZV from cell suspensions that were sonicated before cocultivation with fibroblasts indicated that infectious VZV was not present before reactivation. Moreover, immunohistochemical and immunoprecipitation studies revealed no VZV-specific antigens in any cultures before the reactivation stimulus. VZV antigens were detected after trypsinization and cocultivation. These findings suggest that cultures containing both neurons and satellite cells provide a model system for VZV persistence that possesses many properties of a latent infection.     

人胎脑海马部位神经干细胞形态学观察

目的 探讨人胎脑海马部位神经干细胞的形态学发育特点。方法 收集胎龄20～36周的自愿水囊引产胎儿75例，采用免疫组织化学和光镜观察技术对胎脑海马部位神经干细胞的分布、形态、存在方式进行检测。结果 神经干细胞主要分布于海马多形细胞层、锥体细胞层及颗粒细胞层，分子层也可见，细胞呈圆形、椭圆形、三角形及星形，以圆形细胞多见，0～2个突起不等，以1个突起多见。细胞胞浆丰富；核呈圆形及椭圆形，染色质疏松，2～5个核仁不等。多数单个散在分布，可见对称分裂现象，有的干细胞呈簇状分布，有的干细胞突起与别的细胞保持联系。结论 人胎脑海马部位各层均存在神经干细胞，海马可能存在齿状回之外的神经干细胞生发中心。     

胚胎干细胞诱导分化为造血干细胞重建造血功能的研究

使用胚胎干细胞(embryonic stem cells,ESC)诱导分化为造血干细胞(hematopoieticstem cells,HSC)解决移植供体来源匮乏是目前国际上研究的热点问题.我们使用小鼠ESC体外定向诱导分化为HSC,并移植给骨髓摧毁模型小鼠,观察重建体内造血的情况,借以评价其功能性.     

Effect of hyperoxia on antioxidants in neonatal rat type II cells in vitro and in vivo

Relative resistance to oxygen toxicity in newborn animals (compared to adults) has been associated with increased antioxidant enzymes and glutathione in lung homogenate. The cell type(s) involved in this increase is unknown. We investigated the effect of hyperoxia in vitro and in vivo on the following antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutathione) in alveolar type II cells from neonatal rats. Type II cells were exposed to 95% oxygen or air for 48 h in vitro. When expressed per microgram DNA, all the antioxidants except catalase increased during in vitro incubation; only glucose-6-phosphate dehydrogenase and glutathione increased when expressed per mg protein. None of the antioxidants was higher in oxygen-exposed cells than in air-exposed cells. Neonatal rats were exposed to 100% oxygen or air in vivo for 4 d before determination of antioxidants in lung homogenate supernatant and alveolar type II cells. Catalase, glutathione peroxidase, and glutathione reductase were higher but glucose-6-phosphate dehydrogenase and glutathione were lower in type II cells than in lung homogenate from control animals. Alveolar type II cell glucose-6-phosphate dehydrogenase and glutathione were increased but catalase and glutathione reductase were decreased by exposure to hyperoxia. We conclude that the oxygen-induced increase in whole lung antioxidants is not explained by alveolar type II cell hypertrophy or increased antioxidants within type II cells during hyperoxia.     

The role of fibroblast transdifferentiation in lung epithelial cell proliferation,differentiation, and repair in vitro

Parathyroid hormone-related protein (PTHrP) expression is necessary for differentiation of mesenchymal lipofibroblasts, which induce epithelial type II (TII) cell differentiation, both of which are necessary for alveolarization. PTHrP deficiency may be associated with bronchopulmonary dysplasia (BPD), characterized by truncation of alveolarization among preterm infants. This is supported by the baboon model of BPD (failure of alveolarization) that manifests PTHrP deficiency. We provide evidence that TII cell PTHrP expression is downregulated by alveolar overdistension, resulting in the transdifferentiation of lipofibroblasts to myofibroblasts, characterized by progressive loss of PTHrP receptor expression and triglyceride content, and sequential upregulation of alpha-smooth muscle actin (alphaSMA), typifying fibrosis. PTHrP reverses the downregulation of the PTHrP receptor and upregulation of alphaSMA, reverting myofibroblasts to a lipofibroblast genotype. When TII cells are co-cultured with lipofibroblasts, they proliferate and differentiate, expressing surfactant protein-B; in contrast, TII cells co-cultured with myofibroblasts fail to develop, mimicking the failed alveolarization associated with BPD. Treatment of myofibroblasts with 15-deoxy-Delta 12, 14 prostaglandinJ(2) (PGJ(2)) stimulates ADRP expression, reconstituting the lipofibroblast phenotype. PGJ(2)-treated myofibroblasts promote TII cell growth and surfactant protein-B expression, indicating that failed alveolarization due to transdifferentiation is reversible. We conclude that alveolar overdistension can cause fibroblast transdifferentiation, resulting in failed alveolarization.     

In vitro gene targeting in human Hepatoblastoma

Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches. Steven W. Warmann and Sorin Armeanu contributed equally to this work.