共查询到20条相似文献,搜索用时 31 毫秒
1.
KE Ellison NH Bishopric KA Webster R Morishita GH Gibbons Y Kaneda B Sato VJ Dzau 《Canadian Metallurgical Quarterly》1996,28(7):1385-1399
Current methods of gene transfer into cultured cardiac myocytes have serious limitations, including low efficiency, toxicity or constraints on DNA insert size. The present study examined the effectiveness of hemagglutinating virus of Japan (HVJ) in promoting liposome-mediated DNA transfer into cultured neonatal rat cardiac myocytes. Fluorescein isothiocyanate-labeled oligonucleotides (F-ODN) or plasmid expression vectors encoding SV40 large T antigen (pActSVT) and beta-galactosidase (pAct beta-gal) were complexed with liposomes and the viral protein coat of HVJ. Plasmid vectors were complexed with the nuclear localizing protein HMG-1 prior to HVJ-liposome encapsulation. Neonatal myocytes were transfected by incubation with HVJ-liposome/DNA complexes on culture day 3 or 7. Using F-ODN, we were able to demonstrate significant uptake of DNA (transfection efficiencies of 80-90%) 1 h after transfection that persisted for 1 week in culture. Interestingly, F-ODN were concentrated in the myocyte nuclei for the first 4 days after transfection. Immunohistochemistry showed that 25-30% of myocytes transfected with either pActSVT or pAct beta-Gal expressed plasmid-encoded protein at 72 h whether they were transfected at day 3 or day 7 of culture, while cells transfected with blank vectors did not. Quantitative beta-galactosidase assays confirmed that the use of HVJ significantly enhanced liposome-mediated transfection. Cell toxicity was not apparent. Gene transfer via intracoronary injection also demonstrated the capacity of HVJ to mediate transfection of rabbit cardiac myocytes in vivo, with F-ODN-dependent fluorescence persisting for up to 1 week. We conclude that HVJ/liposome-mediated transfer is efficient for the transfection of both oligonucleotides and plasmids into cardiac myocytes both in vitro and in vivo, and may provide a new tool for the investigation of cardiac myocyte biology and disease. 相似文献
2.
MJ Mann R Morishita GH Gibbons HE von der Leyen VJ Dzau 《Canadian Metallurgical Quarterly》1997,172(1-2):3-12
Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor. 相似文献
3.
HZ Bai Y Sawa WD Zhang T Yamakawa R Morishita Y Kaneda H Matsuda 《Canadian Metallurgical Quarterly》1998,66(3):814-9; discussion 819-20
BACKGROUND: A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ-liposome method. METHODS: The HVJ-liposome complex containing either beta-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate-labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries. RESULTS: In experiment 1, all grafts incubated with beta-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate-labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome-treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ-liposome treatment. CONCLUSIONS: These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ-liposome method and suggest the possibility of its clinical application to prevent vein graft failure. 相似文献
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SI Hayashi R Morishita M Aoki A Moriguchi I Kida M Nakajima Y Kaneda J Higaki T Ogihara 《Canadian Metallurgical Quarterly》1996,3(10):878-885
One of the tools to test an in vitro hypothesis in vivo is transgenic/gene targeting technology. Transgenic technology provides many advantages such as: (1) the study of specific gene function as systemic and developmental effects; and (2) testing of specific gene function chronically. nevertheless, one disadvantage of this technology is the difficulty in tissue-specific targeting of the transgenic expression. Another useful tool is the in vivo gene transfer approach. Therefore, we tested a potential novel model for the study of transgene expression and knock-out using antisense technology. Here, we have demonstrated that incubation of hemagglutinating virus of Japan (HVJ)-liposome complex containing FITC-labeled oligonucleotides in the amniotic fluid of fetal rats resulted in the nuclear localization of fluorescence in the skin (epidermis and dermis). Similarly, transfection of beta-galactosidase gene resulted in positive staining in several surface layers of the skin. Thus, local gene or antisense oligonucleotide transfer approach into the skin may be useful for studying the role of autocrine/paracrine mediators and treating diseases. 相似文献
7.
D Sawamura X Meng S Ina M Sato K Tamai K Hanada I Hashimoto 《Canadian Metallurgical Quarterly》1998,161(10):5633-5639
8.
A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/10(6) cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation. 相似文献
9.
A Bosch PB McCray SM Chang TR Ulich WS Simonet DJ Jolly BL Davidson 《Canadian Metallurgical Quarterly》1996,98(12):2683-2687
Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division. 相似文献
10.
We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma. 相似文献
11.
An inducible bovine KIV* keratin gene promoter was used to target expression of latent or activated transforming growth factor beta 1 (TGF beta 1) to keratinocytes in transgenic mice. This short (2.2-kb) keratin 6 (K6) promoter element was generally silent in untreated animals but was induced in keratinocytes when placed in culture or, in vivo, in response to hyperplasia that follows topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All of the K6-TGF beta 1 transgenic lines studied showed attenuation of the basal keratinocyte proliferative response to 12-O-tetradecanoylphorbol-13-acetate as a consequence of inducible TGF beta 1 gene expression. One of the six lines studied showed constitutive transgene expression at low levels in the skin, and this line had a 2- to 3-fold increase in epidermal DNA labeling index over control mice. Although in vitro TGF beta 1 is known to be a potent negative regulator of epithelial cell proliferation, in vivo TGF beta 1 has complex biological activities and can act as either a positive or negative regulator of keratinocyte proliferation. 相似文献
12.
Y Sawa Y Kaneda HZ Bai K Suzuki J Fujimoto R Morishita H Matsuda 《Canadian Metallurgical Quarterly》1998,5(11):1472-1480
Several of the current techniques for transfer of both oligonucleotide and plasmid DNA into the myocardium are impaired by low efficiency and toxicity. To improve gene transfer techniques, especially into the whole heart, a gene transfer method involving liposome in conjunction with a viral envelope (HVJ-liposome) was essayed as an alternative. FITC-labeled oligonucleotide (F-ODN) and the cDNA of beta-galactosidase (beta-gal) were introduced into the myocardium by coronary infusion of HVJ-liposome during cardioplegic arrest of adult Sprague-Dawley rat hearts. Then, transfected heart was ectopically transplanted into another rat abdomen of the same strain to maintain the transfected heart long enough to allow for protein synthesis. After 3 days of transfection, transfected heart was excised and the efficiency of gene transfection was evaluated. FITC was detected in the nuclei of more than 70% of the myocytes and endothelial cells both in the epicardium and endocardium. beta-Gal was expressed in the cytosol of more than 50% of the myocytes. beta-Gal expression was demonstrated by Western blotting analysis at day 3 after transfection and continued for at least 14 days. No significant histological damage of the myocardium or leakage of CPK were detected in the rats transfected by the HVJ-liposome method. These results clearly demonstrate that the hearts were efficiently transfected both by oligonucleotide and plasmid DNA as a result of coronary infusion of HVJ-liposome during cardioplegic arrest. This thus appears to be an efficient method for gene transfer into the whole heart, providing a new tool for research and therapy for heart diseases. 相似文献
13.
P Assayag H Benamer P Aubry C de Picciotto E Brochet S Besse F Camus 《Canadian Metallurgical Quarterly》1998,82(4):459-464
Most viral vectors are highly immunogenic and are of limited use for somatic gene therapy that requires repetitive administrations. We have developed a highly efficient gene transduction procedure useful for repetitive transfections using liposome containing hemagglutinating virus of Japan (HVJ-liposome). The Escherichia coli beta-galactosidase (beta-gal) gene was embodied in HVJ-liposome, and introduced directly into the caudal lobe of rat liver that was transiently isolated from a systemic circulation. A 116 kDa beta-gal protein was detected in transfected rat liver tissues by Western blot analysis and it was expressed in more than two-thirds of the liver by histological staining. It was found that the transfection efficiency was not affected by repetitive transfections. In support of these findings, antibody response to HVJ-liposome detected in the rat sera was weak and transient. Furthermore, cytotoxic T lymphocytes were not elicited against autologous rat hepatocytes that were transfected in vivo using HVJ-liposome. Thus, our results demonstrate that the isolation of a target liver from systemic circulation and the direct administration of foreign genes using HVJ-liposomes are useful for high gene transduction and persistent gene expression in the liver. 相似文献
14.
N Nakamura K Shino T Natsuume S Horibe N Matsumoto Y Kaneda T Ochi 《Canadian Metallurgical Quarterly》1998,5(9):1165-1170
To define the early biological effect of in vivo introduction of the PDGF-B gene on the healing of ligaments, a HVJ-liposome suspension containing platelet-derived growth factor (PDGF)-B cDNA was injected directly into the injured patellar ligament of 14-week-old male Wistar rats. Rats were killed at 1, 4 and 8 weeks for the morphological analysis of angiogenesis by laminin immunohistochemistry and of collagen deposition by Masson's Trichrome staining and collagen I immunohistochemistry. PDGF-B gene transfer caused the enhanced expression of PDGF in healing ligament up to 4 weeks after transfection, leading to an initial promotion of angiogenesis and subsequent enhanced collagen deposition in the wound. Enhanced and accelerated matrix synthesis in the PDGF-B gene introduced healing ligament suggests that this gene transfer technique may be a potentially useful tool for improving soft tissue repair. 相似文献
15.
The rationale of the somatic gene therapy is the correction of diseases at the most fundamental level. Ideal gene therapy should be achieved by the replacement of the wrong gene sequence of genome with correct one. However, the gene technology to date is yet immature so as to correct the wrong gene sequence in vivo. Potentially, the present technology of gene transfer may provide: 1) correction of cellular dysfunction by expressing the deficient gene; 2) addition of new function for a cell by transferring an exogenous gene; 3) inhibition of unfavorable action of a cell by introducing a counteracting gene. In nephrology, the gene transfer targeted kidney has been challenged at the experimental level. HVJ-liposome method and recombinant adenovirus allow gene transfer to the particular cells in kidney in vivo. Ex vivo gene transfer using mesangial cells and macrophages are another option. Transplant kidney is also a good material for genetic engineering. The potential application of gene transfer is enormous while the therapeutic application have just begun to explored. We have been devoted to HVJ-liposome mediated gene transfer to the kidney and successfully demonstrated the suppression of the extracellular matrix accumulation of the glomeruli in the experimental glomerulonephritis through inhibition of the TGF-beta action by antisense oligonucleotides or soluble type receptor chimera for TGF-beta. We also applied this technology to the inhibition of interstitial fibrosis in unilateral ureter obstruction model. The new HVJ-liposome method improved in lipid composition allows gene transfer to tubulointerstitial fibroblast by retrograde approach from ureter. In consequence, introduced TGF-beta antisense suppressed the TGF-beta mRNA in concomitant with ameliorating interstitial fibrosis. We believe that the gene transfer technique will become common strategy to study the molecular aspect of the renal diseases and will be possibly applicable to molecular intervention in nephrology. 相似文献
16.
L Zhuang B Wang GA Shinder GM Shivji TW Mak DN Sauder 《Canadian Metallurgical Quarterly》1999,162(3):1440-1447
Excess exposure of skin to ultraviolet B (UVB) results in the appearance of so-called sunburn cells. Although it has been demonstrated that sunburn cells represent apoptotic keratinocytes, the molecular mechanisms for UVB-induced apoptosis in keratinocytes have not been fully elucidated. The cytokine, TNF-alpha, has been shown to induce apoptosis in a variety of cell types. Since UVB induces keratinocytes to release TNF-alpha, we hypothesized that TNF-alpha is involved in UVB-induced apoptosis in keratinocytes. In order to confirm this hypothesis and to further delineate which type of TNF receptor signaling mediates the apoptosis pathway, we performed both in vivo and in vitro experiments using gene-targeted knockout mice lacking either the TNF p55 receptor or the TNF p75 receptor. In the in vivo study, wild-type and mutant mice were exposed to UVB, and apoptotic keratinocytes were detected by examining DNA fragmentation using in situ nick-end labeling. For the in vitro experiments, keratinocytes derived from the wild-type and mutant mice were irradiated with UVB, and the degree of apoptosis was determined by flow cytometry, nick-end labeling of DNA, and a DNA ladder assay. Both in vivo and in vitro studies demonstrated that the deletion of TNF receptor p55 could suppress UVB-induced apoptosis in keratinocytes. Our observations support the notion that TNF-alpha is involved in UVB-induced keratinocyte apoptosis, and demonstrate that p55 receptor signaling plays a pivotal role in this event. 相似文献
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For the application of gene therapy to bladder cancer, we examined four in vivo gene transfer methods without viral vectors. For lipofection cationic liposomes (Lipofectin) were instilled into murine bladders. The hemagglutinating virus of Japan (HVJ)-liposomes possessing membrane fusion activity were also injected intraluminally. Using a particle gun, rabbit bladder mucosa was bombarded with DNA-coated gold microcarriers. Electrotransfection was examined in rabbit bladder by pulse direct currents (0.15-0.2 A, 50 msec, repeated 8 times) generated between needle electrodes after submucous injection of DNA solution. beta-galactosidase gene and chloramphenicol acetyltransferase (CAT) gene were used as marker genes. Although lipofection was inefficient in normal urothelium, cancerous urothelium was transfected slightly. HVJ-liposomes more efficiently transfected superficial layers of urothelium with a peak of expression on day 5. The particle gun produced non-uniform but efficient transfection in deeper layers of the urothelium. By electrotransfection, submucous interstitial cells were transfected as well as urothelium. No major complications were observed after these four procedures. HVJ-liposomes are potentially useful for the treatment of carcinoma in situ and the latter two methods may be suitable for the adjuvant therapy of localized bladder tumors. 相似文献
19.
We have studied how keratinocytes cultured under hyperthermal conditions modulate skin fibroblast growth potential and their biosynthetic phenotypes in vitro. When keratinocytes were cultured at 30, 34, 37 or 39 degrees C, the conditioned medium of the keratinocytes cultured at 39 degrees C showed a greater inhibitory activity for fibroblast proliferation and greater synthetic activities of collagen and glycosaminoglycans than those incubated at 30, 34, or 37 degrees C. Transforming growth factor (TGF) beta 1 production in skin fibroblasts was also stimulated by the keratinocyte conditioned medium cultured at 39 degrees C. The stimulating activity of collagen and glycosaminoglycan syntheses of keratinocyte conditioned medium may be explained at least partly by enhanced TGF beta 1 production. The results indicate that keratinocytes cultured at a higher temperature (39 degrees C) may secrete factor(s) which modulate both fibroblast growth and matrix synthesis. This may provide evidence that under hyperthermal conditions epidermis can influence the functions of skin fibroblasts. 相似文献
20.
J Li K Foitzik E Calautti H Baden T Doetschman GP Dotto 《Canadian Metallurgical Quarterly》1999,274(7):4213-4219
We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions. 相似文献