Eukaryotic vectors of Celo avian adenovirus genome,carrying GFP and human IL-2 genes

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.     

过表达核糖核酸酶抑制因子通过调节ILK/PI3K/AKT信号途径抑制小鼠黑色素瘤B16-F10细胞体内外生长

核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞浆内的一种酸性蛋白质.已有研究证明,RI与核糖核酸酶A（RNaseA）和血管生成素（angiogenin,ANG）结合可抑制其活性.本室前期实验证实,RI可有效抑制某些肿瘤的生长和转移. 然而,RI抑制肿瘤的分子机制尚不清楚. 本研究探讨RI对小鼠黑色素瘤B16-F10细胞生长和凋亡的影响及其机制. MTT法结合流式细胞术分析结果证明,RI基因稳定转染导致B16+F10黑色素瘤细胞S期阻滞,抑制B16-F10黑色素瘤细胞增殖. Annexin V/PI结合流式细胞术结果显示,RI过表达引起细胞凋亡.与此相一致,蛋白质印迹分析显示,过表达RI引起抗凋亡分子Bcl-2表达下调,而Bax上调,同时伴有Pro-casepase 3激活. C57BL/ 6小鼠移植成瘤实验显示,与对照相比,转染RI的B16-F10细胞形成的肿瘤重量显著减少,同时伴有肿瘤组织微血管密度降低.提示RI过表达能抑制微血管生成. 此外,体内外组织/细胞免疫化学和蛋白质印迹结果揭示,过表达RI可显著抑制整合素连接激酶(integrin-linked kinase,ILK)下游靶分子Akt和GSK-3β的磷酸化,并降低β-联蛋白的表达.研究结果证明,过表达RI可通过抑制ILK/ PI3K/AKT信号通路,促进细胞凋亡,引起S期阻滞,并抑制血管生成,从而显著抑制小鼠黑色素瘤B16-F10细胞在体内、外的生长.上述结果提示,RI可能是治疗黑色素瘤的有效分子靶点.     

Novel highly sensitive IL-10-beta-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme β-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-β-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.     

A VSV-G pseudotyped HIV vector mediates efficient transduction of human pulmonary artery smooth muscle cells

Attempts were made to infect human vascular smooth muscle cells derived from the pulmonary artery (hPASMC) with two different human immunodeficiency virus (HIV) vector systems. ADA/Luc or HXB2/Luc were generated by cotransfection of luciferase reporter gene vector, pNL4-3-Luc-E- R-, and one of two envelope expressing vectors, pSMADA (R5) or pSMHXB2 (X4). The VSV-G/Luc or VSV-G/GFP were produced by a three-plasmid expression system which consisted of vesicular stomatitis virus G protein (VSV-G) expressing vector, packaging plasmid, and one of two reporter genes (pHR'-CMV-Luc or pHR'-CMV-GFP). We used hPASMC, U87.CD4.CCR5 and U87.CD4.CXCR4 for infection. Neither ADA/Luc nor HXB2/Luc could infect hPASMC, though they could infect U87.CD4 with corresponding coreceptors. On the other hand, the transduction of both VSV-G/Luc and VSV-G/GFP to hPASMC was remarkable. At day 3, the relative proportion of positive cells of hPASMC infected with VSV-G/GFP was 15%. The above finding indicates a direct role of HIV-1 infection in pulmonary hypertension 'a rare complication of HIV-1 infection' and HIV-based vectors could introduce foreign genes into hPASMC for gene therapy of pulmonary hypertension.     

Generation of Pecam1 endothelial specific dual reporter mouse model

Endothelial cells are specialized epithelium lining the interior surface of vessels and play fundamental roles in angiogenesis, vascular permeability, and immune response. To identify endothelial cells in vivo, we constructed a Pecam1^{nlacZ‐H2B‐GFP/+} knock‐in mouse model in which the endothelial cells are labeled by nuclear LacZ (nlacZ) expression. When Pecam1^{nlacZ‐H2B‐GFP/+} mice are bred with germline Cre deleter mice, Pecam1^H2B‐GFP/+ line is created with native nuclear GFP (H2B‐GFP) expression in the endothelium of various organs. This dual reporter mouse provides us with a powerful genetic tool for definitive identification of endothelial cells and monitoring this important cell population throughout development, homeostasis, and disease conditions in mammals.     

Macrosphelide B suppressed metastasis through inhibition of adhesion of sLe(x)/E-selectin molecules

Macrosphelide B (MSB), a 16-membered macrolide from Microsphaeropsis sp. FO-5050, inhibits adhesion of sialyl Lewis(x) (sLe(x))-expressing HL-60 cells to LPS-activated (E-selectin-expressing) human umbilical vein endothelial cells (HUVECs) in vitro. This study examines MSB effects on metastasis of B16/BL6 mouse melanoma cells (B16/BL6 cells) and L5178Y-ML mouse lymphoma cells in vivo and analyzes the MSB antimetastatic activity mechanism. When administered MSB at 20 mg/kg/day, lung metastatic nodules of B16/BL6 cells were significantly decreased (T/C = 45%). However, no inhibition of metastasis of L5178Y-ML cells to the spleen and liver was observed. Flow cytometry analysis showed that B16/BL6 cells expressed high levels of sLe(x) antigen, whereas expression on L5178Y-ML cells was low. Under in vitro conditions, B16/BL6 cells demonstrated a greater degree of adhesion to LPS-activated HUVECs than L5178Y-ML cells, but adhesion was significantly inhibited by MSB and sLe(x) antibody. Combined therapy of MSB and cisplatin (CDDP) induced remarkable lung metastasis inhibition without adverse effects of CDDP to the host. All these findings suggest that MSB suppresses lung metastasis of B16/BL6 cells by inhibiting cell adhesion to endothelial cells through the sLe(x) molecule.     

Dual fluorescent protein reporters for studying cell behaviors in vivo

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color‐coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color‐code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color‐code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time‐lapse movies of fluorescently‐labeled primordial germs cells demonstrate the utility of the color‐code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708–717, 2009. © 2009 Wiley‐Liss, Inc.     

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.     

Developmental regulation of the composite CAG promoter activity in the murine T lymphocyte cell lineage

Promoter selection is of utmost importance for the study of in vivo gene function using transgenic models. In the present study, we have analyzed the expression of the GFP marker under the control of the composite CAG promoter in the lymphoid compartment of several transgenic mouse strains. Despite the ability of the CAG promoter to drive gene expression in almost all tissues examined to date, its activity appears to be developmentally regulated within the T lymphocyte cell lineage. In particular, CD4 and CD8‐expressing, thymic immature T cells displayed lower levels of the GFP marker when compared with both bone marrow precursors and mature circulating T cells, suggesting a transient downregulation of CAG activity during T cell development. Alternative promoters may therefore be preferred for the study of T cell development in vivo using a transgenic approach. genesis 47:799–804, 2009. © 2009 Wiley‐Liss, Inc.     

Immunogenicity of 2 murine B16 melanoma variants with high metastatic potential

The expression of tumor-associated transplantation antigens (TATA) by two metastatic variants, isolated from B16 melanoma in vivo, was examined. The first, YB16 melanoma (amelanotic), was selectioned after a successive s. c. transplantations of B16 melanoma cells on the coisogenic Yellow AY/a mutant mice of C57BL/6J mice. The second, MB16 melanoma, characterized by a variable pigmentation, was obtained from a s. c. transplantation of YB16 melanoma cells on C57BL/6J mice. The comparison of TATA expressed by the two variants and the B16 melanoma, made between different modes of inducing tumor-rejection activity, revealed that i) these two variants failed to induce an autologous antitumor response, ii) they were resistant to crossed immunization with an immunogenic preparations of B16 melanoma and iii) only MB16 melanoma preparations reduced significantly the tumoral incidence of B16 melanoma cells. These data leads us to suggest i) that the s. c. transplantation of B16 melanoma cells on Yellow AY/a mice resulted in the selection of nonimmunogenic, amelanotic and metastatic cell population of YB16 melanoma and ii) the existence of an epigenetic regulation of melanogenesis and expression of TATA in MB16 melanoma cells carried on C57BL/6J mice.     

超正电荷绿荧光蛋白+36GFP作为核酸载体的细胞穿透性分析

旨在通过原核表达纯化超正电荷绿色荧光蛋白+36GFP,研究其与核酸的结合作用及作为核酸载体的细胞转导功能。将pET+36GFP-HA2质粒转化到大肠杆菌BL21(DE3)菌株中,然后表达纯化+36GFP蛋白。将得到的目的蛋白在特定浓度下分别转导293细胞、HepG2细胞、A549细胞和B16细胞,流式细胞仪检测+36GFP的转导效率;+36GFP蛋白(100 nmol/L)转导A549细胞,激光共聚焦显微镜观察结果;将+36GFP蛋白与质粒DNA按不同比例孵育,凝胶阻滞实验检测+36GFP与DNA的结合能力;激光共聚焦显微镜和流式细胞仪检测+36GFP蛋白携带质粒DNA转导细胞后报告基因的表达。结果显示,+36GFP蛋白具有较高的细胞转导效率,且随浓度升高转导效率增加,呈浓度依赖性。凝胶阻滞实验显示,+36GFP能够与质粒DNA结合,阻滞DNA在凝胶中迁移,且呈现一定的浓度依赖性。+36GFP包裹质粒转导细胞后,可高效携带质粒DNA转导进入细胞,使质粒报告基因得到表达。本研究成功表达纯化了+36GFP蛋白,证实该蛋白具有较高的细胞转导效率,可将外源核酸携带入细胞使外源基因得到表达。     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

人胰腺癌细胞再增殖体外模型的建立

目的:细胞再增殖是导致胰腺癌放化疗失败的主要原因之一,但缺乏合适的用于研究胰腺癌再增殖的细胞模型。本研究拟建立简便、实用的胰腺癌细胞再增殖体外模型。方法:表达绿色荧光蛋白-荧光素酶(GFP-Luc)的慢病毒感染人胰腺癌细胞,经嘌呤霉素筛选,用荧光显微镜和流式细胞仪观察双标记细胞GFP表达情况,用生物成像检测双标记细胞Luc活性及分析细胞数量与Luc活性之间的关系。以X-线照射胰腺癌细胞制备饲养细胞,以相应的双标记肿瘤细胞为报告细胞进行共培养。对共培养细胞进行荧光显微镜观察和生物成像,以判断饲养细胞对报告细胞的生长促进作用。结果:通过表达GFP-Luc的慢病毒感染获得双标记人胰腺癌细胞,经荧光显微术、流式细胞术和生物成像术证实这些双标记的人胰腺癌细胞能有效地表达GFP和Luc活性,可作为报告细胞用于建立人胰腺癌再增殖细胞模型。将经X-线照射的饲养细胞和相应的报告细胞共培养,经荧光显微镜观察和生物成像分析,结果显示X-线照射过的饲养细胞对报告细胞的生长具有显著的促进作用。结论:成功建立了简便、实用的人胰腺癌再增殖体外模型,该模型能很好地模拟人体内胰腺癌细胞再增殖过程,为进一步研究胰腺癌细胞再增殖的分子机制提供了新的技术手段。     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。