受四环素调控的真核表达系统SK-Hep1细胞株的建立

建立稳定、高效表达外源基因的SK-Hep1细胞株,以便进一步研究基因的作用.首先将调控质粒pCDNA6/TR转染SK-Hep1细胞,经潮霉素筛选得到多个稳定单克隆.各个单克隆分别扩大培养后,转染pCDNA4/TO/lacZ质粒,再经过DOX(强力霉素)诱导表达,检测β-半乳糖苷酶(β-D galaetosidase,β-gal)活性,从而筛选出高诱导水平低背景表达的SK-Hep1 tet-on细胞株.最后,再将pCDNA4/TO/c-myc质粒转染进SK-Hep1 tet-on细胞株,进一步通过Western blotting检测该系统对下游基因的表达调控.成功建立了一株受DOX调控的高诱导水平低背景表达的细胞株SK-Hep1 tet-on 10#.     

Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

Fusion Protein of Interleukin 4 and Diphtherial Toxin with High Cytotoxicity to Cancer Cells

One means to improve the selectivity of cancer therapyis by directing foreign protein with activity againsttherapeutic targets that displays different expression levelon malignant cells from normal cells. There have hadsubstantial efforts to rationally de…     

NG2-expressing cells in the nervous system revealed by the NG2-EYFP-knockin mouse

The NG2 glycoprotein is a type I membrane protein expressed by immature cells in the developing and adult mouse. NG2+ cells of the embryonic and adult brain have been principally viewed as oligodendrocyte precursor cells but have additionally been considered a fourth glial class. They are likely to be a heterogeneous population. In order to facilitate studies on the function of NG2+ cells and to characterize these cells in situ, we generated an enhanced yellow fluorescent protein (EYFP) "knockin mouse." EYFP-expressing cells in heterozygous knockin mice expressed the NG2 protein in all regions and at all ages studied. The EYFP+ cells did not express markers of mature glia, developing or mature neurons or microglia, but expressed markers typical for immature oligodendrocyte-lineage cells. Examination of the hippocampus showed heterogeneity in the population with regard to expression of S100ss and glutamine synthetase. Furthermore, different subpopulations of NG2+ cells in the hippocampus could be recognized by their electrophysiological properties.     

NG2 cells generate both oligodendrocytes and gray matter astrocytes

NG2 glia constitute a fourth major glial cell type in the mammalian central nervous system (CNS) that is distinct from other cell types. Although circumstantial evidence suggests that some NG2 glia differentiate into oligodendrocytes, their in vivo fate has not been directly examined. We have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express DsRed or Cre specifically in NG2-expressing (NG2+) cells. In NG2DsRedBAC transgenic mice, DsRed was expressed specifically in NG2+ cells throughout the postnatal CNS. When the differentiation potential of NG2+ cells in vitro was examined using DsRed+NG2+ cells purified from perinatal transgenic brains, the majority of the cells either remained as NG2+ cells or differentiated into oligodendrocytes. In addition, DsRed+NG2+ cells also differentiated into astrocytes. The in vivo fate of NG2 glia was examined in mice that were double transgenic for NG2creBAC and the Cre reporter Z/EG. In the double transgenic mice, the Cre reporter EGFP was detected in myelinating oligodendrocytes and in a subpopulation of protoplasmic astrocytes in the gray matter of ventrolateral forebrain but not in fibrous astrocytes of white matter. These observations suggest that NG2+ cells are precursors of oligodendrocytes and some protoplasmic astrocytes in gray matter.     

Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.     

Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells

Receptor of human interleukin 4 (hlL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R,a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.     

Conditional expression of fibroblast growth factor-7 in the developing and mature lung

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.     

Comparison of murine dental follicle precursor and retinal progenitor cells after neural differentiation in vitro

Human dental stem or precursor cells can differentiate into multiple cell types like adipocytes, osteoblasts or chondrocytes. Recently, a number of different human dental stem cell lines were differentiated into neurons. This makes dental stem cells interesting as possible cell-based therapeutics for neural degenerative diseases. To test this hypothesis, we have investigated the neural differentiation potential of murine dental follicle precursor cells (mDFPCs). The mDFPC cell line was newly established without cell immortalization. After differentiation, neural cell marker expression in mDFPCs was checked and compared with that of murine retinal progenitor cells (mRPCs). Differentiated mDFPCs became neuron-like cells with small cell bodies and long/branching neurites, similar to differentiated mRPCs. However, mRPCs showed more complete neural differentiation. Furthermore, 5% of the differentiated mDFPCs and 37% of the differentiated mRPCs were positive for the glia cell marker GFAP (glial fibrillary acidic protein). The data presents new evidence of neural differentiation of mDFPCs, but only a small percentage of mDFPCs differentiated into glia cells, unlike mRPCs.     

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.     

白细胞介素-18基因肌肉注射产生明显抗肿瘤作用(英文)

 为了探讨人白细胞介素 (h IL ) - 1 8基因转导的抗肿瘤作用 ,构建了 h IL- 1 8c DNA真核表达质粒载体 pc DNA3/h IL- 1 8.将 pc DNA3/h IL- 1 8经脂质体包裹 ,按照 1 0 0μg/只的剂量注射入雄性Balb/c小鼠后肢肌肉 ,在设定时间处死小鼠取注射部位肉提取总 RNA,应用半定量反转录聚合酶链反应 (RT- PCR)及免疫组化法检测了 h IL- 1 8m RNA及其蛋白质在小鼠肌肉中不同时间点的表达水平 .随后 30只雄性 Balb/c小鼠于基因注射后第 7d腹部皮下 (i.d.)或腹腔内 (i.p.)接种 1×1 0 5个同系小鼠 S- 1 80肉瘤细胞 .观察了 S- 1 80肿瘤细胞在腹腔及皮下的生长情况、小鼠体重、肿瘤重量及存活时间 .结果发现 ,pc DNA3/h IL- 1 8注射后 2 d能检测到 h IL- 1 8m RNA表达 ,第 7d表达量较高 ,第 2 8d仍有 h IL- 1 8m RNA表达 .免疫组化染色显示了注射部位散在有 h IL- 1 8蛋白质颗粒 .h IL- 1 8基因注射组小鼠体重、肿瘤重量均比对照组轻 (P值分别小于 0 .0 0 5、0 .0 5) ,存活时间比对照组延长 .结果显示 ,真核表达系统 pc DNA3/h IL - 1 8经脂质体包裹肌注后能有效表达 ,具有明显的抑制肿瘤生长作用 .     

Polydendrocytes display large lineage plasticity following focal cerebral ischemia

Polydendrocytes (also known as NG2 glial cells) constitute a fourth major glial cell type in the adult mammalian central nervous system (CNS) that is distinct from other cell types. Although much evidence suggests that these cells are multipotent in vitro, their differentiation potential in vivo under physiological or pathophysiological conditions is still controversial.To follow the fate of polydendrocytes after CNS pathology, permanent middle cerebral artery occlusion (MCAo), a commonly used model of focal cerebral ischemia, was carried out on adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in polydendrocytes and their progeny. The phenotype of the EGFP(+) cells was analyzed using immunohistochemistry and the patch-clamp technique 3, 7 and 14 days after MCAo. In sham-operated mice (control), EGFP(+) cells in the cortex expressed protein markers and displayed electrophysiological properties of polydendrocytes and oligodendrocytes. We did not detect any co-labeling of EGFP with neuronal, microglial or astroglial markers in this region, thus proving polydendrocyte unipotent differentiation potential under physiological conditions. Three days after MCAo the number of EGFP(+) cells in the gliotic tissue dramatically increased when compared to control animals, and these cells displayed properties of proliferating cells. However, in later phases after MCAo a large subpopulation of EGFP(+) cells expressed protein markers and electrophysiological properties of astrocytes that contribute to the formation of glial scar. Importantly, some EGFP(+) cells displayed membrane properties typical for neural precursor cells, and moreover these cells expressed doublecortin (DCX)--a marker of newly-derived neuronal cells. Taken together, our data indicate that polydendrocytes in the dorsal cortex display multipotent differentiation potential after focal ischemia.     

人宫颈癌细胞Hela移植模型肿瘤生长的荧光分析和卡尺测量的比较

目的建立稳定表达绿色荧光蛋白的人宫颈癌细胞系,建立移植瘤模型并比较移植模型肿瘤生长的荧光分析和卡尺测量的优缺点。方法以Lipofectamine 2000介导chickenβ-actin-GFP-NEO转染人宫颈癌细胞Hela,经梯度浓度G418筛选获得稳定表达绿色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,利用活体荧光成像系统和游标卡尺观察肿瘤的生长情况。结果获得了稳定表达GFP的人宫颈癌细胞株,将其接种到裸鼠体内可成瘤。活体荧光成像观察发现,1至3周随着肿瘤体积逐渐增大,平均荧光光子数逐渐增加;4周时随着肿瘤出现明显坏死,平均荧光光子数呈现下降趋势,而游标卡尺测量结果显示肿瘤在4至5周仍然不断的增大。结论绿色荧光蛋白能够在人宫颈癌细胞Hela中长期稳定表达,用绿色荧光蛋白标记的人宫颈癌细胞Hela建立的裸鼠肿瘤模型可以为人宫颈癌研究提供理想的实验材料,应用小动物活体成像系统能够客观定量评价活的肿瘤细胞在动物体内的生长情况,而不是肿瘤体积的变化。     

Effect of NRG1, GDNF,EGF and NGF in the Migration of a Schwann Cell Precursor Line

The Schwann cells are the myelinating glia of the peripheral nervous system that originated during development from the highly motile neural crest. However, we do not know what the guidance signals for the Schwann cell precursors are. Therefore, we set to test some of the known neurotrophins that are expressed early in developing embryos and have been shown to be critical for the survival and patterning of developing glia and neurons. The goal of this study was to determine more specifically if GDNF, NRG1 and NGF are chemoattractants and/or chemokinetic molecules for a Schwann cell precursor line, the Spl201. We performed live chemoattraction assays, with imaging and also presented these molecules as part of their growing substrate. Our results show for the first time that GDNF and NRG1 are potent chemoattractive and chemokinetic molecules for these cells while NGF is a chemokinetic molecule stimulating their motility.     

Establishment of a New Human Glioblastoma Multiforme Cell Line (WJ1) and Its Partial Characterization

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.     

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

     

Reconstitution of nude mouse T cell function in vivo: IL 2-independent effect of human T cells

The i.p. injection of 1 to 5 X 10(6) heavily irradiated human T lymphocytes resulted in the lasting reconstitution of T cell functions in young mice bearing the nu/nu mutation. IgM and IgG responses to immunization with sheep red cells or ovalbumin, splenic lectin responses, and the expression of easily detectable Thy-1 determinants on up to 20% of spleen cells could be documented for several months after the injection of human cells. Only a narrow cell dose range was effective. Injection of larger cell numbers not only failed to induce immune reconstitution but also resulted in the development of resistance to subsequent treatments. Only mature T cells, but not thymocytes, could induce nude mouse T cell development. Lymphoblasts from one patient with acute T cell leukemia consistently immune-reconstituted nude recipients. These cells were completely unable to produce IL 2 in vitro. In contrast, the IL 2-producing T cell line Jurkat was ineffective, indicating that the abilities to produce IL 2 and to induce nude mouse T cell development are independent. In an extension of earlier models of the nude mouse immune defect, two distinct T precursor cell pools are proposed as the major components of an extrathymic differentiation pathway. As an adequate trigger of differentiation, interaction with thymus-processed T cells guides the development of precursors in the first cell pool towards populating the second IL 2-responsive pool of T precursor cells.