Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F2alpha secretion by luminal epithelial, glandular epithelial and stromal cells from pig endometrium. II. Responses of cyclic, pregnant and pseudopregnant pigs on days 12 and 16 post oestrus

In pigs, the exact mechanism for the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine mode during pregnancy recognition is not known. The objective of this study was to examine whether this shift involved a change in the responsiveness of luminal epithelial, glandular epithelial and stromal cells to 0 or 100 nM oxytocin. Luminal epithelial cells, glandular epithelial cells and stromal cells were isolated from cyclic, pregnant or oestrogen-induced pseudopregnant gilts on Day 12 (Experiment 1) or Day 16 (Experiment 2) post oestrus (oestrus = Day 0). For cells obtained on Day 12, oxytocin stimulated PGF2alpha secretion by stromal cells (P<0.01) similarly for each reproductive status, whereas oxytocin stimulated PGF2alpha secretion from luminal and glandular epithelial cells (P<0.05) from pregnant and pseudopregnant gilts but not from cyclic gilts. For both concentrations of oxytocin, mean PGF2alpha secretion was less (P<0.05) from stromal cells of pregnant than cyclic gilts. For cells obtained on Day 16, oxytocin stimulated PGF2alpha release from stromal cells of cyclic gilts but not from stromal cells of pregnant gilts. Mean PGF2alpha secretion also was less (P<0.05) from stromal cells of pregnant gilts than cyclic gilts. Oxytocin tended to stimulate PGF2alpha release (P<0.07) from glandular epithelial cells of cyclic but not pregnant or pseudopregnant gilts. Luminal epithelial cells from all reproductive statuses were similarly unresponsive to oxytocin. In conclusion, the increased PGF2alpha secretory response to oxytocin of luminal and glandular epithelial cells from pregnant gilts on Day 12, combined with the decreased response of stromal cells from pregnant gilts on Days 12 and 16, may contribute, in part, to the shift in endometrial PGF2alpha secretion from an endocrine to an exocrine direction during early pregnancy in pigs.     

低剂量辐射对脐血LAK细胞体外抗肿瘤活性的影响

目的 探讨低剂量辐射对脐血LAK细胞体外抗肿瘤活性的影响。方法 新鲜分离的脐血单个核细胞用重组白细胞介素2培养72h获取LAK效应细胞,在培养48h时给予1次不同剂量（分别为62mGy,124mGy,186mGy,248mGy）的γ射线照射,应用^3H－TdR释放实验测定LAK体外杀伤肿瘤靶细胞（K562,HL－60）活性。结果 脐血LAK细胞对K562和HL－60的杀伤活性与成人外周血相比差异无显著性（P＞0．05）。接受低剂量辐射的脐血LAK细胞K562和HL－60的细胞毒性显著高于非照射对照组（P＜0．01）。结论 脐血可作为LAK细胞的一个良好来源。低剂量辐射可增强脐血LAK杀伤肿瘤细胞的细胞毒性,从而可能用于清除微小残留病变及在脐血移植时增强移植物抗白血病效应。     

三叶青提取物对白血病HL60细胞增殖抑制作用研究

目的探讨三叶青提取物对白血病HL60细胞的增殖、凋亡的影响。方法将不同浓度的受试物作用于体外培养的HL60细胞,用四氮唑蓝比色法（MTT）检测细胞生长抑制率,应用流式细胞仪观察细胞凋亡。结果受试物处理24 h后6.25、12.50、25.00、50.00、100.00μg/m l组,受试物处理48、72 h后所有浓度组HL60细胞的增殖率和凋亡率均呈不同程度的下降或上升,差异均有统计学意义。结论三叶青提取物能有效抑制体外HL60细胞的增殖,诱导HL60细胞凋亡。     

人宫颈癌干细胞的分离鉴定

目的:通过分选人宫颈癌细胞中的侧群细胞验证其肿瘤干细胞特性。方法:取16例行宫颈鳞癌切除的手术标本进行宫颈癌细胞原代培养,流式细胞仪分选侧群细胞(Side Population,SP),对比SP细胞和非SP细胞(Non Side Population,Non-SP)的体外自我更新能力和肿瘤形成能力。结果:在原代培养的16株宫颈癌细胞中均分选出SP细胞,比例约占(1.67±0.94)%。SP细胞的克隆形成能力显著高于NSP细胞,SP细胞在裸鼠肿瘤形成能力显著高于NSP细胞。结论:人宫颈癌侧群细胞具有肿瘤干细胞特性,为宫颈癌干细胞的研究提供了简单、有效的分离方法。     

Nutrient utilization by cells isolated from rat jejunum, cecum and colon

Cells isolated from the jejunum, cecum and colon of rats were used to study the oxidation of nutrients by quantifying the production of 14CO2 from 5 mmols/L 14C-labeled exogenous substrate. In colonic cells, the decreasing order of oxidation was as follows: butyrate greater than acetate greater than propionate, glucose and glutamine. Acetate and butyrate significantly suppressed oxidation of both glucose and glutamine. In cells taken from the cecum, butyrate was oxidized at a greater rate than all other substrates. Cells taken from the jejunum produced CO2 from exogenous substrates in decreasing order as follows: glutamine greater than glucose much greater than acetate, propionate and butyrate. Butyrate oxidation was significantly reduced in colonic cells by 3-hydroxybutyrate, and it was reduced in cecal cells by glucose. Comparisons among the three gut segments showed no differences in glutamine oxidation. Glucose oxidation was greater in cells taken from the colon than from the cecum or jejunum, which were similar. Butyrate and acetate were oxidized at higher rates in cells taken from the cecum and colon than in cells taken from the jejunum, and propionate was oxidized at a greater rate in cells taken from the colon than from the jejunum. These studies demonstrate that relative rates of substrate oxidation differ along the intestinal tract of rats.     

Copper uptake and retention in liver parenchymal cells isolated from nutritionally copper-deficient rats

Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (less than 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver, anemia, low plasma ceruloplasmin oxidase activity and increased 64Cu whole-body retention. Freshly isolated liver parenchymal cells from copper-deficient rats showed a higher 64Cu influx, which was associated with a higher apparent Vmax of 45 +/- 4 pmol Cu.mg protein-1.min-1 as compared with 30 +/- 3 pmol Cu.mg protein-1.min-1 for cells isolated from copper-sufficient rats. No significant difference in the apparent Km (approximately 30 mumol/L) was observed. Relative 64Cu efflux from cells from copper-deficient rats was significantly smaller than the efflux from cells from copper-sufficient rats after prelabeling as determined by 2-h efflux experiments. Analysis of the medium after efflux from cells from copper-deficient rats showed elevated protein-associated 64Cu, suggesting a higher incorporation of radioactive copper during metalloprotein synthesis. Effects of copper deficiency persist in primary cultures of parenchymal cells derived from copper-deficient rats, and short-term cultures of these cells offer a prospect for the study of cell biological aspects of the metabolic adaptation of the liver to copper deficiency.     

2,3,7,8-tetrachlorodibenzo-p-dioxin selectively enhances spontaneous IgE production in B cells from atopic patients

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on spontaneous IgE production was studied in B cells from atopic patients with allergic rhinitis, atopic eczema/dermatitis syndrome or bronchial asthma, and from non-atopic subjects. TCDD enhanced spontaneous IgE production in B cells from atopic patients without affecting production of IgG1, IgG2, IgG3, IgG4, IgM, IgA1 and IgA2, while TCDD failed to induce IgE production in B cells from non-atopic subjects. Purified surface IgE+(sIgE+) B cells from atopic patients spontaneously produced IgE, while surface IgE- (sIgE−) B cells failed to do so. TCDD enhanced spontaneous IgE production in sIgE+B cells, while TCDD with or without IL-4 or anti-CD40 mAb failed to induce IgE production in sIgE−B cells. Collectively, TCDD selectively enhanced ongoing IgE production. These results suggest that TCDD may aggravate allergic diseases by enhancing IgE-mediated allergic responses.     

Irradiation effect on osteoclastogenesis stimulated by breast cancer cells

To examine the effects of carbon ion and gamma ray irradiation on cancer-induced osteoclastogenesis, mouse calvaria MC3T3-E1 cells were cultured with conditioned medium from irradiated and non-irradiated MCF7 human breast cancer cells. The authors examined RANKL and OPG mRNA expression in osteoblastic MC3T3-E1 cells following treatment with conditioned MCF7 medium. Co-cultured MC3T3-E1 and bone marrow cells treated with conditioned medium from irradiated MCF7 cells showed decreased numbers of osteoclasts, assessed using TRAP staining. Conditioned medium from control MCF7 cells elevated the RANKL/OPG mRNA ratio in MC3T3-E1 cells; this effect was suppressed by carbon ion irradiation of the MCF7 cells. These data demonstrate that indirect interactions between breast cancer cells and MC3T3-E1 cells induce osteoclastogenesis in vitro through modulation of RANKL expression and that this process is suppressed by carbon ion irradiation.     

The distribution of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its effect on the vitamin A content in parenchymal and stellate cells of rat liver

Isolated liver cells from male Sprague-Dawley rats given a single dose of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms, 0.9 microCi/kg body wt in corn oil, p.o.) or vehicle only were separated into parenchymal and nonparenchymal cell fractions 4 h, and 1, 4, 7, 25, 50, and 147 d after treatment. Vitamin A content and TCDD-derived radioactivity were estimated in the parenchymal cells and in the stellate cells, which were identified and quantified in these fractions. Similar levels of vitamin A (0.3 +/- 0.4 nmol per million cells or 0.5 +/- 0.7 mumol per liver; values are mean +/- SD for 56 rats) were found in parenchymal cells from both control and TCDD-treated rats. However, while the vitamin A content of stellate cells increased from 14 to 46 nmol per million cells (i.e., from 1.7 to 7.7 mumol per liver) in control rats over the course of the study, stellate cells from TCDD-exposed rats showed no increase in vitamin A level until at least 25 d after exposure and remained at a level about 30% below the controls thereafter. TCDD-derived radioactivity resided mainly in the parenchymal cell compartment, although stellate cells contained more radioactivity per cell. Most of the radioactivity in parenchymal cells was eliminated with a half-life of 13 d, whereas the remainder persisted with an elimination half-life of 70 d. The elimination half-life in stellate cells was estimated to be 52 d. Thus, TCDD inhibited storage of vitamin A in stellate cells until 60-90% of the TCDD-derived radioactivity had been eliminated from the liver.     

Prospects for cell-based therapies for liver disease

Liver parenchymal maintenance and regeneration after injury are physiologically supported by 3 cell compartments: mature liver cells, intra-organ stem cells such as cells of the proximal biliary tree and periductal cells, and extra-organ stem cells from the circulation and the bone marrow. In the latter case, hepatocyte derivation from circulating cells (plasticity) can arise via direct transdifferentiation (site specific, receptor/ligand dependent) or by fusion of circulating cells with pre-existing hepatocytes. Other non-physiologic stem cells, such as mesenchymal stem cells from the bone marrow and embryonic stem cells, may be potentially used in treatment of inherited and acquired liver diseases. This review updates our current understanding of these various cell populations and of possible approaches to their future therapeutic uses in cell transplantation, bioartificial liver devices, cytokine/chemokines manipulation of physiological repair pathways, and gene therapy.     

Toxic effects of Kintoki bean (Phaseolus vulgaris) lectin on cultured animal cells

Effects of a toxic lectin from Kintoki beans (Phaseolus vulgaris, cultivar, Kintoki) on four types of animal cells were investigated. The cells used in this study were mouse L 929, human HeLa S3, and tendon and liver cells from chick embryo. The Kintoki bean lectin agglutinated these cells not only in suspension but also in monolayer, resulting in the marked growth inhibition of these cells. The incorporations of 3H-thymidine and 3H-leucine into trichloroacetic acid insoluble fraction of the cells were considerably inhibited by this lectin. There was, however, some lag period before the revelation of the inhibition. Kintoki bean lectin bound to these cells within 1 hr. The type of the lectin receptor seemed uniform for each cell type and the number of the binding sites per cell was different from cell type to cell type. When Kintoki bean lectin was removed from the culture medium, these cells slowly recovered back to normal growth.     

黑米花色苷对血管内皮细胞过氧化损伤的保护作用

目的:探讨黑米花色苷对ox-LDL引起的血管内皮细胞损伤的保护作用。方法:分别在4种黑米花色苷预孵保护作用下用ox-LDL过氧化处理EVC304细胞株,通过加载GIF滤光片的倒置显微镜观察细胞形态;通过MTT实验,荧光分光光度法和2’,7’-二氯荧光素双醋酸盐(DCFH-DA)荧光探针分别检测细胞活力及体内丙二醛和自由基变化;用流式细胞仪分析细胞周期。结果:四种黑米花色苷能明显减轻ox-LDL对内皮细胞形态的损伤,降低ox-LDL对细胞增殖的抑制作用,显著减少细胞内丙二醛增加量;其中,矢车菊素-3-葡萄糖苷还能显著降低ox-LDL所致自由基的生成量,促进细胞由G1期进入S期,促进细胞增殖,减少细胞凋亡。结论:黑米花色苷对ox-LDL引起的血管内皮细胞过氧化损伤有明显保护作用。     

The role of interferon in the NK cell killing of virus-infected target cells.

Spleen cells from uninfected CBA mice are more cytotoxic for Sendai virus-infected L929 cells than for uninfected cells and the lymphocytes responsible have the properties of NK cells. Preincubation of spleen cells with culture supernatants from Sendai virus-infected L929 cells increases the cytotoxicity for uninfected target cells. This increase in cytotoxicity can also be produced by pretreatment with purified mouse interferon. The enhancing effect of both the infected culture supernatants and purified interferon can be neutralized with anti-interferon serum. It is concluded that the preferential killing of Sendai virus-infected L929 cells by NK cells is dependent on the induction of interferon and that interferon will increase NK cell cytotoxicity for uninfected target cells.     

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.     

Differentiation of embryonic and adult stem cells into insulin producing cells

Replacement of insulin producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is successful in experienced centers. The wider application of this therapy, however, is limited by the lack of donor organs. Insulin producing cells generated from stem cells represent an attractive alternative. Stem cells with the potential to differentiate into insulin producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns but research with human ESC may help us to decipher important steps in the differentiation process in vitro since almost all information available on pancreas development are based on animal studies. The present review summarizes the current knowledge on the development of insulin producing cells from embryonic and adult stem cells with special emphasis on pancreatic, hepatic and human mesenchymal stem cells.     

Metabolism of 1,2-dimethylhydrazine by cultured rat colon epithelial cells

The colon carcinogen 1,2-dimethylhydrazine (DMH) was cultured with rat colon epithelial cells to determine if these cells have the ability to metabolize DMH. Colon epithelial cells isolated from conventional and germfree Sprague-Dawley rats were incubated in CMRL 1066 supplemented medium containing 14C-DMH. Cells from both groups of rats metabolized DMH to gaseous metabolites, to metabolites in the medium that were putatively identified as azoxymethane and methylazoxymethanol, and to products that bound to DNA. Cells from germfree rats metabolized DMH at an equal or greater rate than cells from conventional rats for the criteria examined. This report demonstrates that rat colon epithelial cells can metabolize DMH without previous metabolism by other tissues or colon bacteria.     

Maintenance of pluripotential embryonic stem cells by stem cell selection

As gastrulation proceeds, pluripotential stem cells with the capacity to contribute to all primary germ layers disappear from the mammalian embryo. The extinction of pluripotency also occurs during the formation of embryoid bodies from embryonic stem (ES) cells. In this report we show that if the initial differentiated progeny are removed from ES cell aggregates, further differentiation does not proceed and the stem cell population persists and expands. Significantly, the presence of even minor populations of differentiated cells lead to the complete loss of stem cells from the cultures. This finding implies that the normal elimination of pluripotent cells is dictated by inductive signals provided by differentiated progeny. We have exploited this observation to develop a strategy for the isolation of pluripotential cells. This approach, termed stem cell selection, may have widespread applicability to the derivation and propagation of stem cells.     

An effective method for recovering CD34 positive progenitor cells from peripheral blood stem cell apheresis products cryopreserved with simplified method

The use of small volume collection chamber (SVCC) during peripheral blood stem cell (PBSC) apheresis, combined with simplified cryopreservation without rate-controlled freezing, have successfully been applied to clinical PBSC transplantation following myeloablative chemotherapies. However, the method to effectively select CD34+ progenitor cells from frozen apheresis products obtained with these simplified methods has not been reported. For this goal, after washing the thawed apheresis products with medium containing Dnase I, two different approaches to purify CD34+ progenitor cells from washed WBCs were compared. In method I, CD34+ cells were purified on the same day using immunomagnetic method. In method II, the cells after wash were cultured for overnight in RPMI-1640/10% FCS containing SCF and IL-3, followed by enrichment of CD34+ cells as in method I on the next day. In both methods, CD34+ cells were recovered with high viability. However, subsequent liquid culture revealed that the cells obtained from method II have clearly higher growth potential compared with the cells from method I. In addition, these CD34+ cells from method II well-tolerated to further refreeze and thaw. Thus, allowing to "rest" overnight after thaw may be critical for processing of the simply cryopreserved apheresis products as in method II.     

Immunobiology of dendritic cells and their application in clinical practice

INTRODUCTION: Dendritic cells play a central role in the regulation of the immune responses towards cellular or humoral immunity. Several types of dendritic cells can be distinguished, which originate from different hematopoietic lineages, and differ from each other in function, morphology and localization within the organism. Immature dendritic cell-precursors originate from bone marrow haemopoietic stem cells, circulate in blood system and reach peripheral tissues, where they differentiate into active antigen capturing and processing cells. They have further maturation by the effect of adequate signals (antigen stimuli and inflammatory cytokines) and migrate into secondary lymphoid organs, presented cell surface MHC molecules-linked peptides from antigens which they have captured and processed, to T lymphocytes, induce immune reaction by T cell activation. Apart from the fact that dendritic cells as professional antigen presenting cells are essential to induction T cell-mediated immune responses, they influence activation and function of B lymphocytes and effector cells of natural immune system by cytokines and cell-cell interactions. CONCLUSIONS: Complex regulatory function of dendritic cells make a possibility for artificial alteration of immune processes by modification of dendritic cells: direct against definitive antigens, increase or decrease their intensity, hereby we can accomplish the immunotherapy of different diseases such as tumors, infection, autoimmune diseases, transplants' rejection.     

Cells adherent to copper-bearing intrauterine contraceptive devices determined by monoclonal antibodies

The presence of nucleated cells adherent to copper-bearing IUDs removed from successful IUD users, as well as from those IUD users with accidental pregnancy, was determined by a panel of monoclonal antibodies. A significant decrease in the percentage of CD3+ cells-mature T lymphocytes was found in the cell population adherent to IUDs removed from pregnant compared to non-pregnant uteri (43 +/- 2.6 vs 34 +/- 1.5). Among these cells, the percentage of CD4+ cells was increased (from 22.9 +/- 1.9 to 30.4 +/- 2.0), and CD8+ was decreased (from 23 +/- 0.9 to 10.8 +/- 1.2). The percentage of HLA-DR+ cells was also decreased (from 24.3 +/- 1.7 to 16.7 +/- 1.8). B4+ cells (B lymphocytes) were present in a similar percentage on IUDs removed from pregnant as well as from non-pregnant uteri. Thus, the uterine cavity in the presence of an IUD, contains a consistent population of immunologically competent cells. The question still remains, whether the change in the number of nucleated cells present in the uterine cavity with an IUD could contribute to its antifertility effect.