人白血病抑制因子在昆虫细胞Sf9中的表达

人白血病抑制因子(hLIF)cDNA装入p2bac,受其多角蛋白启动子控制,并与野生型线性杆状病毒DNA共转染昆虫细胞Sf9。经ELISA和免疫印迹证实,该重组病毒感染Sf9 24h后(胞解液)和48h后(培养液),均可测得表达的hLIF,在72h时蛋白浓度可达每毫升(1×10~7细胞)4～10μg;经细胞活性观察表明,该蛋白可促进人白血病细胞U937分化,并使U937内信号分子STAT_3合成增加。结果表明,昆虫细胞表达的hLIF可分泌于培养液中且含量高。它的高表达、易纯化、强活性,有实用价值。     

Retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells is potentiated by leukemia inhibitory factor

Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III beta-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes -fetoprotein and GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.     

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.     

Gene expression in human neural stem cells: effects of leukemia inhibitory factor

Human neural precursor cells grown in culture provide a source of tissue for drug screening, developmental studies and cell therapy. However, mechanisms underlying their growth and differentiation are poorly understood. We show that epidermal growth factor (EGF) responsive precursors derived from the developing human cortex undergo senescence after 30-40 population doublings. Leukemia inhibitory factor (LIF) increased overall expansion rates, prevented senescence and allowed the growth of a long-term self renewing neural stem cell (ltNSCctx) for up to 110 population doublings. We established basal gene expression in ltNSCctx using Affymetrix oligonucleotide microarrays that delineated specific members of important growth factor and signaling families consistently expressed across three separate lines. Following LIF withdrawal, 200 genes showed significant decreases. Protein analysis confirmed LIF-regulated expression of glial fibrillary acidic protein, CD44, and major histocompatibility complex I. This study provides the first molecular profile of human ltNSCctx cultures capable of long-term self renewal, and reveals specific sets of genes that are directly or indirectly regulated by LIF.     

Temporal and spatial expression of leukemia inhibitory factor in rabbit uterus during early pregnancy

Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley-Liss, Inc.     

Birth control vaccine targeting leukemia inhibitory factor

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin‐6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF‐receptor (LIF‐R) that span the regions involved in ligand‐receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF‐R‐mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long‐lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF‐R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF‐R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF‐R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF‐R is also important for implantation and pregnancy in women. Mol. Reprod. Dev. 79:97–106, 2012. © 2011 Wiley Periodicals, Inc.     

Pulmonary expression of leukemia inhibitory factor induces B cell hyperplasia and confers protection in hyperoxia

Leukemia inhibitory factor (LIF) is produced by a large number of pulmonary cells in response to diverse stimuli. Exaggerated levels of LIF have also been detected in the adult respiratory distress syndrome and other disorders. The biologic effects of LIF in the lung, however, have not been elucidated. To define the respiratory effects of LIF, we generated transgenic mice in which human LIF was selectively targeted to the mature lung. In these mice, transgene activation caused an impressive increase in bronchoalveolar lavage (BAL) cellularity with a significant increase in BAL and tissue B lymphocytes. LIF also conferred protection in 100% O2 where it decreased alveolar-capillary protein leak and enhanced survival. This protective effect was associated with the induction of interleukin (IL)-6 mRNA and protein. LIF transgenic mice with a null mutation in IL-6 were more sensitive to the toxic effects of 100% O2 than LIF-transgenic animals with a wild-type IL-6 locus. These studies demonstrate that LIF induces B cell hyperplasia and confers protection in hyperoxic acute lung injury. They also demonstrate that LIF induces IL-6 and that the protective effects of LIF are mediated, in part, via this inductive event. LIF may be an important regulator of B cell-mediated responses and oxidant injury in the lung.     

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.     

Retinoic acid alters EGF receptor expression during palatogenesis

Various growth factors are necessary for normal embryonic development and EGF receptors are present in developing palatal shelves of embryonic/fetal mice at least from day 12 of gestation. The medial epithelium of the palatal shelf undergoes a series of developmental events which do not occur in the oral and nasal epithelia. In utero and in organ culture, the control palatal medial epithelium shows a developmental decline in EGF receptors, demonstrated both by a decrease in the binding of antibody to EGF receptors and a decrease in the binding of 125I-EGF; decreases which are not observed in cells of the adjacent oral or nasal epithelium. During this period, medial cells cease DNA synthesis and undergo programmed cell death. Medial epithelial cells exposed to all-trans-retinoic acid continue to express EGF receptors, bind EGF, proliferate, fail to undergo programmed cell death and exhibit a morphology typical of nasal cells. The data suggest that this disturbance by retinoic acid of EGF receptor localization and subsequent alterations in differentiation of the epithelial cells plays a role in the retinoic-acid-mediated induction of cleft palate.     

Tumor necrosis factor inhibits K+ current expression in cultured oligodendrocytes

Summary The effects of tumor necrosis factor- (TNF-), a cytokine secreted by activated macrophages, on the electrical membrane properties of cultured adult ovine oligodendrocytes (OLGs) were investigated using the whole-cell voltage-clamp technique. Treatment with recombinant human TNF- (rhTNF) for 24 to 72 hr produces (i) process retraction in some but not all OLGs, (ii) a reduction in the resting membrane potential with no significant change in membrane capacitance or input resistance over control cells and (iii) a decrease in the expression of both the inwardly rectifying and outward K⁺ current. The magnitude of the membrane potential change as well as K⁺ current inhibition was larger in cells with retracted processes. The electrophysiological effects of rhTNF were attenuated when rhTNF was neutralized with a polyclonal anti-rhTNF antibody. The binding of rhTNF to its receptor has been reported to increase GTP binding, to increase GTPase activity of a pertussis-sensitive G protein, and to produce an elevation in intracellular cAMP in other cell types. However, pretreatment of OLGs with activated pertussis toxin failed to attenuate or mimic the effects of rhTNF. Chronic exposure of OLGs to the membrane permeant analogue of cAMP, 8-bromo-cAMP, resulted primarily in an inhibition of the inwardly rectifying K⁺ current, an effect which was less than that produced by rhTNF alone and without any of the associated rhTNF-induced morphological changes. This indicates that the effects of rhTNF cannot be entirely accounted for by an elevation in intracellular cAMP. Cycloheximide (CHX), an inhibitor of protein synthesis, mimicked the effects of rhTNF; however, the effects of rhTNF and CHX were not additive. The finding that both ionic current expression and membrane potential were reduced in cells treated with rhTNF that appeared morphologically normal suggests that abnormal ion channel expression in OLGs precedes and may contribute to eventual myelin swelling and damage.     

Association of increased bcl-2 expression with rescue from tumor necrosis factor-alpha-induced cell death in the oligodendrocyte cell line OLN-93

The present study investigated the effects of flupirtine (Katadolon) on tumor necrosis factor (TNF)-alpha-mediated cell death and Bcl-2 expression in the permanent rat oligodendrocyte cell line OLN-93 (OLN cells). TNF-alpha (500 U/ml) induced apoptosis of OLN cells, which was confirmed by DNA fragmentation using an in situ end-labeling technique and ultrastructural analysis. Flupirtine significantly reduced the rate of spontaneous cell death of OLN cells already at low concentrations; TNF-alpha-mediated apoptosis was suppressed only with higher concentrations of flupirtine (100 microM:). Expression of Bcl-2 protein and mRNA in OLN cells was detected by immunocytochemistry, western blot, and RT-PCR. Quantitative analysis of western blots revealed an approximately 2. 5-fold up-regulation of Bcl-2 protein during TNF-alpha treatment. Furthermore, addition of 10 or 100 microM: flupirtine before incubation with TNF-alpha led to an approximately threefold increase of Bcl-2 expression. Exposure of OLN cells to flupirtine alone moderately augmented the expression of Bcl-2 protein. Our data demonstrate that flupirtine up-regulates the expression of Bcl-2 protein in OLN cells; this Bcl-2 induction is associated with a reduced rate of TNF-alpha-induced cell death.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.