nm23-H1基因转染对人胆管癌细胞系QBC939体外浸润能力的影响

目的：探讨nm23-H1基因转染对人胆管癌细胞系QBC939体外浸润能力的影响。方法：将含有全长nm23-H1 cDNA的真核表达载体通过脂腩体法转染人胆管癌细胞系。结果：转染成功的QBC939细胞，其nm23-Hl基因的mRNA、蛋白表达明显增加，转染nm23-H1基因的胆管癌细胞体外浸润能力下降，穿越matrigel的细胞数明显低于亲本QBC939细胞，代表浸润能力的IV型胶原酶（MMP-9）分泌量下降。结论：nm23-Hl基因可以抑制胆管癌细胞的体外浸润能力。     

Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives

Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.     

人酪氨酸羟化酶基因真核表达载体的构建及在哺乳动物细胞中的表达

用限制性内切酶EcoRI从pKS(-)HTH_1切下全长为1.9 kb的人酪氨酸羟化酶基因,在T_4DNA连接酶的作用下连接在真核表达载体pCDNA_3的EcoR Ⅰ位点,构建成重组质粒pcD-NA_3HTH_1,该质粒转染COS-7细胞,免疫荧光组织化学染色证实酪氨酸羟化酶在其中的表达。     

Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells

A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared to cells transfected with expression-vector alone or untransfected cells. However, when injected subcutaneously into nude mice, both NCAM expressing cells and control cells produced invasive tumors. Nude mice injected with NCAM positive cells developed tumors with slower growth rates as compared to those induced by NCAM negative cells. This implies that NCAM may not only be involved in adhesive and motile behaviour of glioma cells, but also in their growth regulation.     

Sequences in the proximal 5′ flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.     

Rb基因对人视网膜母细胞瘤株SO-Rb50的影响

目的：探讨视网膜母细胞瘤（Rb）的发生与Rb基因（Rb1）缺失、失活等异常的关系。方法：用逆转录病毒载体pDOR与全长4．7kb野生型Rb1cDNA构建逆转录病毒表达载体pDOR－Rb1+。用脂质体介导法将pDOR－Rb1+转入CRIP包装细胞系。结果：实验产生了0．5X105Cfu具有一次感染能力的重组逆转病毒。利用该病毒感染SO-Rb50，经G418筛选，获得了抗G418细胞群体。运用PCR及Southern杂交技术对转染细胞进行检测，结果表明该抗G418细胞中有完整的外源Rb1存在。Northern杂交发现其Rb1mRNA表达水平有所提高。对细胞群体生长速率、软琼脂集落形成能力的测定表明，外源Rb1的表达使SO-Rb50在软琼脂中集落形成能力降低，而群体生长速率无明显影响。结论：外源Rb1对SO-Rb50恶性表型有一定影响。     

Stable Transfection of Nonosteogenic Cell Lines with Tissue Nonspecific Alkaline Phosphatase Enhances Mineral Deposition Both in the Presence and Absence of β-Glycerophosphate: Possible Role for Alkaline Phosphatase in Pathological Mineralization

It is documented that alkaline phosphatase (AP) plays an important role in bone mineralization. Considering that TN-AP is expressed in periodontal ligament fibroblasts, renal epithelial cells, and vascular endothelial cells, and that TN-AP is both a calcium-/phosphate-binding protein and a phosphohydrolytic enzyme, we hypothesize that membrane-bound AP also plays an important role in the initiation of physiological and pathological mineralizations in tissues other than bone and cartilage. To test this hypothesis, nonosteoblast cell lines, including a fibroblast line, a renal epithelial line, and a capillary endothelial line, were stably transfected to express high levels of rat bone AP on their cell surfaces. These rat bone AP-expressing cells were then cultured on filter membranes in the presence or absence of β-glycerol phosphate. von Kossa staining for calcium phosphate and transmission electron microscopy with electron diffraction analysis for minerals were employed to investigate the effect of membrane AP on extracellular calcium phosphate mineralization. Our results indicated that AP expression on these nonosteoblast-like cell surfaces have induced extracellular hydroxyapatite (HAP) mineralization. Our findings support the concept that membrane-bound AP contributes to extracellular apatitic mineralization by mechanisms that do not necessarily involve its hydrolase activity. They also suggest that AP might be important for the initiation of pathological mineralization in nonosteogenic tissues. Received: 11 January 1996 / Accepted: 31 October 1996     

胞嘧啶脱氨酶基因修饰神经干细胞及其表达的离体实验研究

目的 探讨胞嘧啶脱氨酶(cytimidine deaminase，CD)基因修饰神经干细胞及其基因表达。方法 通过构建真核表达质粒pCMVCD，限制性内切酶消化鉴定后，采用Lipofectamine 2000脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stem cells，NSCs)，G418筛选阳性克隆，加入不同浓度的5-氟胞嘧啶(5-Flourocytosine，5-FC)，MTT比色法测定NSCs的生存率。结果 本实验成功地培养并鉴定了神经干细胞，并将CD基因成功地转染了神经干细胞，G418阳性NSCs对低浓度5-FC高度敏感。结论 CD基因修饰神经干细胞的离体实验研究为干细胞治疗研究提供依据。     

Construction of Antisense Transforming Growth Factorβ_1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

Summary:To construct the antisense transforming growth factorβ1(TGFβ1)gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells.TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells(MG-63)and inserted into pcDNA_3 to con-struct an antisense expression vector,which was dubbed pcDNA_3-TGFβ1(-).MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene.Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly.It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation,which mightbe helpful for gene therapy of osteosarcoma.