MK──一种新发现的细胞因子

MK是一种新发现的细胞因子,属于肝素结合因子家族,MK是一种小分子多肽,其基因表达仅见于胚胎中期及成年期肾脏,在许多肿瘤细胞中也可见MK基因不同程度的表达,MK能够促进正常细胞的生长和分化,特别是促进神经细胞的发育,它还可以抑制某些肿瘤细胞的生长．MK基因在成年肾脏中表达的原因尚未阐明．     

中因子与人类疾病

中因子(Midkine,MK)是神经营养性并受发育调节的肝素结合性生长因子家族的成员之一。该家族包括中因子、多向促激素(pleiotrophin),以及鸟类中因子相似物RI-HK,MK看起来在妊娠中期胚胎发生中起重要作用。在包括某些癌症和阿尔茨海默(Alzheimer's)病的病理情况下,MK被不适当地表达。 MK是一种14kDa的肝素结合性生长因子。这是一种高碱性的糖基化多肽,具有五个链内二     

LIM蛋白KyoT在小鼠胚胎发育中的表达

LIM结构域蛋白KyoT可与转录因子RBP-J相互作用而修饰Notch信号途径.以往发现KyoT在成年小鼠肺脏、肾脏和睾丸中有特异性表达,为进一步明确KyoT在小鼠胚胎阶段的表达,故分析了KyoT在小鼠不同胚胎阶段的表达水平变化和胚胎17天时各组织的表达分布.采用RNA印迹发现KyoT在小鼠胚胎的各阶段均表达,而且胚胎17天时表达水平最高.进一步RNA印迹和免疫组织化学检测发现,KyoT mRNA和蛋白质在胚胎17天小鼠的肺、肾以及肌肉中均有高水平的表达.上述结果提示,KyoT在小鼠胚胎发育过程中有特异性表达,且在胚胎17天时KyoT表达器官分布与成年小鼠相似,特异性定位于肺、肾和肌肉等脏器.     

中期因子在肿瘤发生和组织再生中的作用

中期因子(midkine,MK）是一种肝素结合性生长因子.在胚胎期,MK在组织中广泛分布.在成人体内其表达降低,仅局限于某些特定部位.MK受体种类繁多,信号通路复杂多样,这就决定了MK功能的多样化,它能促进很多种类细胞的生长、存活、分化和迁移,具有抗细胞凋亡的作用,不仅与肿瘤发生密切相关,而且在很多组织的发育形成及损伤后的修复再生过程均有参与.MK已成为恶性肿瘤在内的多种疾病治疗中颇具前景的分子靶点.本文中对MK的基因及蛋白结构、受体及相关信号通路、分子功能及作用机制等进行了全面的综述,并对其在肿瘤发生和发育与组织再生等方面的生物学功能及研究意义进行了深入的探讨.     

滇龙胆甲羟戊酸激酶基因的克隆与表达分析

甲羟戊酸激酶是甲羟戊酸途径的关键酶。根据滇龙胆转录组甲羟戊酸激酶基因Gr MK序列,设计一对基因特异性引物克隆该基因,并进行序列分析;构建原核表达载体p GEX-4T-1-Gr MK,转入大肠杆菌Rosetta(DE3),重组蛋白在37℃、1.0 mmol/L IPTG诱导下成功表达。生物信息学分析结果表明,Gr MK蛋白为甲羟戊酸激酶家族成员,具有MK蛋白保守结构域:乳糖激酶/高丝氨酸激酶/甲羟戊酸激酶/磷酸甲羟戊酸激酶(GHMP kinase)N端结构域、C端结构域和ATP结合结构域;Gr MK与长春花Cr MK亲缘关系最近.原核表达结果表明,融合蛋白相对分子质量与推断大小一致。组织特异性表达分析结果表明Gr MK基因主要在根中表达.这些结果为Gr MK蛋白结构和功能的研究奠定基础。     

尼古丁影响小鼠胚胎发育的分子机制初探

目的:探讨不同剂量的尼古丁对小鼠胚胎组织器官发育的影响及其分子机制。方法:以妊娠期雌鼠为评价模型,通过皮下注射不同浓度的尼古丁,在E18.5时解剖获取胚胎并称重,同时采集各组织器官,应用HE染色观察相关组织的病理学变化,并采用qRT-PCR检测相关基因表达水平的变化。结果:与对照组相比,尼古丁对小鼠E18.5胚胎的脑、肺、肝脏、肾脏有明显损伤;qRT-PCR分析显示血管形成相关基因(VEGF-α)、神经生长相关基因(Egr-1)及细胞生长分化相关调控基因(c-FOS)有显著的表达差异,其中VEGF-α在心脏和肾脏中表达下调,Egr-1在肺和肾脏中表达上调,c-FOS在脑、心脏和肺中表达上调。结论:妊娠期母鼠注射尼古丁会对小鼠胚胎的脑、肺、肝脏、肾脏产生明显损伤,并导致VEGF-a、Egr-1及c-FOS基因表达异常。     

MK基因对小鼠子宫基质细胞蜕膜化进程的影响

为探索肿瘤坏死因子相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)的死亡受体(mouse killer,MK)对小鼠子宫基质细胞蜕膜化进程的影响,构建MK基因过表达和siRNA干扰重组腺病毒.原代培养的小鼠子宫基质细胞感染MK过表达或者干扰重组腺病毒并诱导蜕膜化,72 h后用免疫细胞化学与流式细胞术分别检测蜕膜细胞的标志物催乳素(prolactin,PRL)与蜕膜细胞凋亡率的变化情况.妊娠d4小鼠子宫角注射MK重组腺病毒,观察胚胎植入点的数量变化.实验结果表明,与对照组相比,在诱导的蜕膜细胞中过表达MK使得催乳素的含量显著降低(P<0.05),同时,蜕膜细胞的凋亡率明显升高(P<0.05),而siRNA干扰之后催乳素的含量显著升高,凋亡率明显下降(P<0.05),但是,宫角注射MK基因过表达和siRNA干扰重组腺病毒之后,胚胎植入数量均显著减少(P<0.01).提示MK基因通过参与小鼠子宫内膜基质细胞的蜕膜化进程,调节蜕膜细胞增殖与凋亡之间的平衡从而影响胚胎的植入.     

小鼠胚胎对人卵巢癌细胞生物学行为影响的体外研究

探讨体外共培养环境中小鼠胚胎对人卵巢癌细胞HO8910PM的影响.通过小鼠胚胎与肿瘤细胞体外共培养模型观察小鼠胚胎对肿瘤细胞的形态及生长行为的影响,Annexin V-EGFP/PI原位检测与小鼠胚胎共培养后肿瘤细胞的凋亡情况,MTT粘附实验、transwell迁移及侵袭实验研究与小鼠胚胎共培养后的人卵巢癌细胞的粘附性、迁移性及侵袭性的变化.共培养时小鼠胚胎能够侵入人卵巢癌细胞并推开肿瘤细胞形成自己的生长空间,激光共聚焦显微镜下见胚胎周围的肿瘤细胞凋亡增加,与对照组比较共培养后的HO8910PM肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P0.05、P0.01).结果表明体外共培养体系中小鼠胚胎能够侵袭肿瘤细胞,促进人卵巢癌细胞的凋亡,并使其粘附性、迁移及侵袭相关恶性行为降低.     

骨髓间充质干细胞和部分肿瘤细胞中Nucleostemin基因的表达

以分离的人胚胎和大鼠骨髓间充质干细胞 (MSCs) ,6种肿瘤细胞株 ,裸鼠肿瘤和转移瘤组织为实验材料 ,以大鼠心肌组织和人胎盘组织为对照 ,探讨nucleostemin基因的表达情况 .RT PCR结果显示 ,nucleostemin基因在MSCs、肿瘤细胞和肿瘤组织中均有不同程度的表达 ,而大鼠心肌和人胎盘组织中无表达 .DNA测序结果证明 ,扩增的PCR产物与GenBank提供的DNA序列完全同源 .SCID裸鼠肿瘤动物模型定量PCR结果证实 ,nucleostemin的mRNA在裸鼠肿瘤组织和转移瘤组织中表达较高 .研究结果表明 ,在细胞中nucleostemin基因不同水平的表达可能与MSCs、肿瘤细胞的增殖和肿瘤的发生、发展与转移有关 .     

UBC家族新成员UBF基因的组织表达谱研究

目的和方法:提取人胎肝和HL-60细胞总RNA,采用RT-PCR的方法,从中扩增UBF基因,并对扩增产物进行测序,从而证实UBF基因的天然存在性;采用原位杂交的方法研究UBF在5月龄胎儿的不同组织及HL-60细胞中的表达谱及亚细胞定位.结果:从胎肝和HL-60细胞中均扩增得到了完整的UBF基因,且其序列与注册序列完全一致;原位杂交结果显示UBF在人胚胎多种组织中表达,其中胎骨骼肌表达最强,胎肾最弱.结论:UBF基因,作为Ubc家族的一个新成员,在人胚胎的骨骼肌、肝脏、肾脏、心脏和肺中均有表达,且骨骼肌中的表达最强.     

Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene,which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1

MK gene was intensely expressed, when aggregates of HM-1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM-1 cells correlated with the concentration of retinoic acid, especially between 10^-8 and 10^-7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis

Midkine (MK) is the first cloned gene in a new family of heparin- binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.     

Regulation of Osteoblast Gene Expression in Intratypic Osteosarcoma Hybrid Cells

Intratypic osteosarcoma hybrids were constructed by fusing the human osteoblast-like osteosarcoma SaOS-2 with the rat osteoblast-like osteosarcoma UMR-106. Both of these osteosarcomas express liver/bone/kidney alkaline phosphatase (ALPL), but only the UMR-106 cell line expresses osteopontin (OPN), a gene expressed during later stages of osteoblast differentiation. Analysis of osteoblast gene expression in these hybrids demonstrated that ALPL continued to be expressed; however, OPN steady-state mRNA levels were dramatically reduced in four hybrids. Quantitative measurements indicated theft OPN steady-state mRNA levels were extinguished by a factor of 20- to 1000-fold. Since SaOS-2 chromosomes are preferentially lost from these hybrids, subclones of extinguished hybrids were isolated that reexpressed OPN mRNA at levels similar to the UMR-106 parental line. These data indicate that trans-acting negative regulatory factors, expressed from the SaOS-2 genome, are responsible for OPN extinction. This report provides the first demonstration of the negative regulation of OPN gene expression and also provides additional evidence that extinction plays a role in the regulation of osteoblast gene expression.     

Cloning of HumanENC-1and Evaluation of Its Expression and Regulation in Nervous System Tumors

We recently identified and characterized a novel murine gene,ENC-1,that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role forENC-1gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue ofENC-1.The humanENC-1gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels ofENC-1mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of theENC-1gene during neural crest cell differentiation. We found that the expression ofENC-1increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest thatENC-1expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.     

Forced Expression of the Homeobox-Containing Gene Pem Blocks Differentiation of Embryonic Stem Cells

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous;Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.