阳离子脂质体转染人类骨骼肌原代细胞的初步研究

探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体.     

Efficient cold transfection of pea ferredoxin-NADP(H) oxidoreductase into rat hepatocytes

We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non-transfected cells, the engraftment of FNR-positive cells was not associated with inflammatory reaction. The percentage of FNR-positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of beta-galactosidase-positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4-transfected hepatocytes on engraftment in the recipient parenchyma.     

Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Anti-HIF-1α-pEGFP重组质粒的构建及表达

目的构建EGFP-HIF-1α反义重组质粒,转染人宫颈癌Hela细胞,用以探讨HIF-1α蛋白在宫颈癌的生长、转移中的作用。方法应用基因重组技术构建EGFP-HIF-1α反义重组质粒,通过脂质体介导将其转染入Hela细胞,倒置荧光显微镜观察转染效果,Western blot检测HIF-1α蛋白的表达。结果酶切鉴定结果显示含EGFP-HIF-1α反义重组质粒构建成功,并能在Hela细胞中封闭HIF-1α蛋白的表达。结果 EGFP-HIF-1α反义重组质粒构建及转染成功,封闭Hela细胞中HIF-1α蛋白的表达,为进一步研究HIF-1α蛋白在宫颈癌的生长、转移中的作用提供试验基础。     

Enhancement of DNA uptake in FUT8‐deleted CHO cells for transient production of afucosylated antibodies

Removal of the core α1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody‐dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins. FUT8KO cells evaluated in PEI transfections yielded lower titers than parental CHO WT cells. FACS and HPLC analyses revealed that the FUT8KO cells had lower cell surface heparan sulfate (HS) levels than CHO WT. Removal of cell surface HS resulted in reduced uptake of PEI‐transfected DNA (PEI:DNA) and lower transfection titers suggesting that PEI:DNA relies on HS for binding and cellular entry. The absence of cell surface HS did not severely impact transfections performed with cationic liposomes. We undertook two approaches to improve transient production of afucosylated antibodies. First, we evaluated transfection of FUT8KO cells with cationic liposomes, which were observed to be less dependent on HS levels for uptake. Transfection of FUT8KO cells using the cationic liposome, DMRIE‐C, produced similar titers to CHO WT in both shake flask and large‐scale 10 L bioreactors. The second approach was to engineer a cell line overexpressing exostosin‐1 (EXT1), an enzyme responsible for HS chain elongation, to increase HS content. EXT1‐FUT8KO and CHO WT cells produced comparable levels of antibody from PEI transfections. Biotechnol. Bioeng. 2010;106: 751–763. © 2010 Wiley Periodicals, Inc.     

Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus

Five cell lines (LJHK, LJS, LJL, LJH-1 and LJH-2) were established from the head kidney, spleen, liver and heart of sea perch Lateolabrax japonicus . The cell lines LJHK, LJS, LJL, LJH-1 and LJH-2 were subcultured 46, 32, 32, 36 and 34 times in minimum essential medium (MEM) supplemented with foetal bovine serum (FBS), sea perch serum and 10 ng ml⁻¹ basic fibroblast growth factor (bFGF). Morphology of primary cultures and subcultures of the five cell lines were observed continuously by microscopy. The suitable temperature for growth was 18 to 30° C for all of these cell lines with the optimum growth at 24° C and a reduced growth rate <18° C. The optimum concentration of FBS was found to be 10% and addition of bFGF to the medium significantly increased the growth rate of the cells. The doubling time of LJS, LJH-1, LJL, LJH-2 and LJHK cells was determined to be 52·7, 54·9, 57, 58·7 and 66 h at a plating density of 1 × 10⁵ cells ml⁻¹ at 24° C, respectively. Chromosome analysis revealed that 42, 48, 38, 43 and 45% cells maintained normal diploid chromosome number (48) in the LJH-1, LJH-2, LJHK, LJL and LJS cell lines, respectively. The LJHK cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. Furthermore, treatment of the LJHK cells with lipopolysaccharide led to increased expression of IL-1β, demonstrating that LJHK cells might be a valuable tool for studying the expression and function of immunomodulatory gene in fishes.     

外源基因转染导致小鼠体细胞过快衰老 及p16INK4a表达水平变化

对小鼠胎儿成纤维细胞进行外源基因转染时发现, 外源基因转染后的小鼠体细胞染色体端粒的长度以每代47 bp碱基缩短; 在转染后的衰老细胞中, 或细胞随着增龄, p16^INK4a 5′-调控区DNA甲基化程度逐渐降低; 利用RT-PCR与Northern blot证明, 衰老细胞与年轻细胞中的p16^INK4a基因的表达水平存在显著差异, 传代45代的细胞和外源基因转染后的衰老细胞p16^INK4a基因的表达水平大约是原代细胞的12~16倍, 而原代细胞与20代细胞间的差异很小。外源基因转染后的衰老细胞核移植后能支持克隆胚胎的体外早期发育。     

Immuno-gene therapy of cancer with tumour-mRNA transfected dendritic cells

We have developed immuno-gene therapy for malignant melanoma and prostate cancer. The therapy is based on monocyte-derived dendritic cells (DCs) that are transfected with autologous melanoma-mRNA or mRNA from three prostate cancer cell lines (DU-145, LN-CaP and PC-3). A broad spectrum of tumour-associated antigens will be included in both DC-vaccines. The use of autologous melanoma-mRNA moreover allows targeting of individual tumour antigens that are specific to each patient. Effective protocols have been established for mRNA-transfection by square wave electroporation and for the generation of clinical grade DCs. A full scale preclinical evaluation demonstrated in vitro T cell responses in 6/6 advanced melanoma patients. The responses were specific to antigens encoded by the transfected tumour-mRNA. Recently, we have conducted two phase I/II trials, in advanced malignant melanoma and androgen-resistant prostate cancer. Successful vaccine preparations were obtained for all 41 patients elected. No serious adverse effects were observed. Specific T cell responses (T cell proliferation and/or IFNγ ELISPOT) were demonstrated in 9/19 evaluable melanoma patients and in 12/19 prostate cancer patients. The response rates were higher for patients receiving intradermal vaccination, compared to intranodal injection. Thirteen prostate cancer patients developed a decrease in log-slope PSA. The PSA-response was significantly related to the T cell response (P=0.002). We conclude that the DC-vaccine is feasible and safe, and that T cell responses are elicited in about 50% of patients.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.     

Growth suppression induced by Notch1 activation involves Wnt-beta-catenin down-regulation in human tongue carcinoma cells

BACKGROUND INFORMATION: Involvement of Notch1 signalling in several cancers is well known, but its role in human tongue squamous cell carcinoma, one of the most common carcinomas of the human oral cavity, remains poorly characterized. RESULTS: Our studies demonstrated that constitutively over-expressed active Notch1, via stable transfection of exogenous ICN (intracellular fragment of Notch), resulted in growth suppression of the human tongue cancer cell line Tca8113 in vitro and in vivo, accompanied by G(0)-G(1) cell cycle arrest and apoptosis. Moreover, down-regulation of beta-catenin protein expression was observed in Tca8113 cells stably expressing active Notch1. Activated Notch1 also led to dramatic increase in p21(WAF1/CIP1) and p53 expression with decreases in Skp2 (S-phase kinase-associated protein 2) and Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) expression, which may participate in the induction of apoptosis and cell cycle arrest. CONCLUSIONS: Since the effects of the Notch1 pathway are cell-type specific and context-dependent in cell types where Notch1 has an anti-proliferative effect, down-regulation of Wnt/beta-catenin signalling may be one of the mechanisms which induces apoptosis and cell cycle arrest.     

Scalable production of adeno-associated virus type 2 vectors via suspension transfection

Vectors derived from adeno-associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno-associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large-scale suspension culture could replace these multiple culture preparations, but there is currently no effective co-transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel-coated six-well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 10(13) rAAV particles and, more importantly, up to 10(11) infectious particles were generated from a 2-L bioreactor culture. The suspension-transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor.