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排序方式: 共有216条查询结果,搜索用时 31 毫秒
1.
Herbert Radner Yosuf El-Shabrawi Robert H. Eibl Oliver Brüstle Lukas Kenner Paul Kleihues Otmar D. Wiestler 《Acta neuropathologica》1993,86(5):456-465
Introduction into fetal rat brain cells of a replication-defective retroviral vector harboring v-Ha-ras and v-gag-myc rapidly causes the induction of highly malignant undifferentiated neuroectodermal tumors following transplantation into the brains of syngeneic hosts [Wiestler, et al. (1992) Cancer Res. 52: 3760–3767]. In the present study, we have investigated the modulating effect of the developmental stage of neural target cells and of the dose of the retroviral vector used in the grafting experiments. Exposure of fetal cells from embryonic day (E)12 or E14 produced a 100% incidence of malignant neuroectodermal tumors which led to the death of recipient animals after a median latency period of 32 days. A 100-fold reduction of the virus dose from 2.062×106 to 2.062×104 focus-forming units/ml resulted in a lower tumor incidence of 25%. Of six neural grafts exposed to v-Ha-ras and v-myc at E16, only one showed evidence of tumorigenesis (low-grade astrocytoma and hemangioma). All other transplants were morphologically normal for observation periods of 26 weeks, indicating a marked loss of transforming activity of ras and myc in more advanced stages of brain development. In retrovirus-exposed donor cells which caused the development of neural tumors in recipient rats, malignant transformation was also evident during culture in vitro, usually after 9–12 days. Oncogene complementation was also studied in the newborn rat brain. After microinjection of the retroviral vector into the brain at postnatal day (P)0, P1 and P3, 5 out of 20 animals (25%) developed a total of seven brain tumors. Histopathologically, three of these neoplasms were malignant neuroectodermal tumors which, in contrast to those induced in fetal brain transplants showed evidence of focal glial and/or neuronal differentiation. In addition, we observed one oligodendroglioma, two hemangiomas and a malignant hemangioendothelioma. These data indicate that neural precursor cells and endothelia of the rat brain represent the major target cells for the complementary action of ras and myc and that the use of target cells from later developmental stages (E16 and postnatal) leads to the induction of both primitive and more differentiated neoplasms.These studies were supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich (Erwin Schrödinger fellowship, JO501-MED), by the Swiss National Science Foundation and by the Cancer League of the Kanton of Zürich 相似文献
2.
Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer 总被引:6,自引:0,他引:6
Scholten KB Schreurs MW Ruizendaal JJ Kueter EW Kramer D Veenbergen S Meijer CJ Hooijberg E 《Clinical immunology (Orlando, Fla.)》2005,114(2):119-129
Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies. 相似文献
3.
将人白细胞介素2(hIL-2)cDNA克隆到逆转录病毒载体MNSM的PL位点 ,分别构建了转录受SV40早期启动子和人甲胎蛋白增强子调控的重组逆转录病毒载体MNSI和MNSIA。用脂质体转染法将MNSI和MNSIA分别转导PA317包装细胞,测质粒转染率为(5~20)×10~(-3)克隆/μgDNA·10~6细胞,病毒感染率为(5.4~450)×10~4CFU/ml。重组病毒在4μg/ml polybrene存在条件下感染人肝癌细胞、肾癌细胞和黑色素瘤细胞,Neo~R克隆经Southernblot分析证明hIL-2cDNA转入人肿瘤细胞并整合, R NA斑点杂交及IL-2活性表达分析证明,人甲胎蛋白增强子可促进异源启动子启动hIL-2cD-NA在合成甲胎蛋白的人肝癌细胞中高效特异转录和表达。该研究对肝癌特异性免疫增强基因治疗有重要意义。 相似文献
4.
Specific cytotoxic T lymphocytes in gene therapy 总被引:1,自引:0,他引:1
U. Altenschmidt Dirk Moritz B. Groner 《Journal of molecular medicine (Berlin, Germany)》1997,75(4):259-266
Cytotoxic T lymphocytes possess the capacity to lyse target cells which express antigens on their surface recognized by the
T cell receptor. These cells are crucial in the body’s defense against foreign antigens. It has long been a goal of tumor
biology to utilize T cells, specialized in the elimination of unwanted cells, for the treatment of cancer. The killing activity
of T lymphocytes is restricted to specific antigen-presenting cells. For this reason the use of cytotoxic T cells in the elimination
of cancer cells is limited to cancer cells which present neoantigens on their surface. To circumvent this limitation we describe
a procedure in which the ζ component of the T cell receptor is genetically manipulated and equipped with an extracellular
recognition domain. Introduction of a chimeric gene, consisting of the ζ chain of the T cell receptor and a single-chain antibody
domain, into cytotoxic T lymphocytes results in T cells with a predetermined recognition specificity for particular tumor
cells. The MHC restriction of target cell recognition can be avoided and tumor cells recognized by the single chain antibody
domain can be recognized and lysed. Retroviral-mediated gene transduction was used to introduce chimeric ζ chain constructs
into primary T cells of mice. The cocultivation of retrovirus producing helper cells with in vitro activated T lymphocytes
led to a high gene transduction efficiency into primary T cells. These primary T cells assumed a predetermined specificity
for target cell recognition and lysis. The production and provision of tumor cell specific T lymphocytes might not be sufficient
to eradicate large tumors in vivo. Using a Schwannoma cell line, we showed that transplanted tumors secrete transforming growth
factor β and thereby stifle the action of lymphocytes. We suggest that a coordinated strategy including the suppression of
tumor cells specific antilymphocyte action and the provision of tumor cell specific T cells might be required to successfully
eliminate tumor cells in vivo.
Received: 13 September 1996 / Accepted: 30 October 1996 相似文献
5.
Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a higher copy number of transgenes in keratinocytes and also a higher metabolic capacity. 相似文献
6.
目的探讨HSV-TK/GCV自杀基因系统对人宫颈癌细胞系Hela体外及体内杀伤作用及其产生的旁观者效应.方法采用脂质体转染法将GINaTK载体转入包装细胞PA317.取病毒上清液感染人宫颈癌细胞Hela,得到带有HSV-TK基因的Hela/TK细胞,并将其分别用于体外和体内实验.结果载体HSV-TK导入了PA317细胞.体外实验结果显示,当Hela/TK细胞数占混合细胞10%时,低浓度(10μg/ml)的GCV就可将50%左右的肿瘤细胞杀死.体内实验结果显示GCV可明显抑制Hela/TK细胞在BALB/C小鼠体内的肿瘤形成.经GCV治疗后,肿瘤体积分别较对照组肿瘤体积缩小约11.1%、30.6%和47.2%(均P<0.001);RT-PCR检测HSV-TK基因在肿瘤组织中有表达;实验组肿瘤组织与对照组相比存在明显的病理学改变.结论逆转录病毒可介导HSV-TK基因转入人宫颈癌细胞Hela并获稳定表达,HSV-TK/GCV自杀基因系统在体内外对宫颈癌细胞均有杀伤作用,且存在明显的旁观者效应. 相似文献
7.
M. E. Verberne A. C. Gittenberger-de Groot R. E. Poelmann 《Anatomy and embryology》1998,198(3):171-184
We were interested in the contribution of the cardiac neural crest to the complete anterior and posterior nerve plexus of
the chick heart. This includes the pathways by which these cardiac neural crest-derived neuronal precursors enter the heart.
As lineage techniques we used the traditional quail-chick chimera in combination with the newly introduced technique of retroviral
reporter gene transfer to premigratory cardiac neural crest cells. Retrovirally infected embryos (n=23) and quail-chick chimeras (n=19) between stages HH27 and 40, were immunohistochemically evaluated, using the lineage markers LacZ (retroviral reporter)
and QCPN (anti-quail nuclear marker), respectively and the neuronal differentiation markers HNK-1, RMO-270 and DO-170. Between
stages HH27 and 33, quail-derived and LacZ positive cells were situated around the arterial cardiac vagal branches at the
arterial pole, and vagal branches along the anterior cardinal veins and the sinal vagal branch at the venous pole. From stage
HH35 onward, QCPN/LacZ-positive cardiac ganglia were observed throughout the anterior and posterior plexus and were mainly
concentrated in the subepicardium near the distal ends of the arterial cardiac vagal branches and the sinal cardiac vagal
branch respectively. From stage HH36 both the anterior and posterior plexus contained a population of large cardiac ganglion
cells and a population of smaller cells along nerve branches as well as in the cardiac ganglia, which means that differentiation
starts in both plexus at the same time. Furthermore only nerve fiber connections between the anterior and posterior plexus
were observed. These results show that the cardiac neural crest contributes to the cardiac ganglion cells from both the entire
anterior and posterior plexus. Furthermore these results suggest that these precursor cells enter the arterial pole via the
arterial cardiac vagal branches and the venous pole via the sinal cardiac vagal branch without intermixing. Finally we show
that in addition to the cardiac ganglia, the cardiac neural crest contributes to small myocardial glia or undifferentiated
cells along nerve fibers, and some myocardial nerve fibers as well as nerve tissue in the adventitia of the large veins at
the venous pole and in the adventitia of the coronary arteries.
Accepted: 30 March 1998 相似文献
8.
逆转录病毒载体介导乙型肝炎病毒反义基因的转录表达 总被引:2,自引:0,他引:2
为了探索在真核细胞内转录表达乙型肝炎病毒(HBV)反义核酸的方法,用基因重组技术将HBV前C/C基因(PreC/C)和前S/S基因(PreS/S)片段反向插入逆转录病毒载体质粒,再将重组体分别转染PA317包装细胞,进而获得能够介导HBV反义基因向小鼠NIH3T3细胞转移表达的重组逆转录病毒。经分子杂交试验表明,含有HBV反义基因的重组逆转录病毒序列已经整合到转染的PA317细胞染色体上;转导的NIH3T3细胞内有HBV反义RNA转录表达。结论:逆转录病毒载体包装细胞系统能够介导HBV反义基因在真核细胞中转录表达,因而有可能利用反义技术和基因转移方法进行抗-HBV基因治疗 相似文献
9.
为了寻找和研究新的人类基因cDNA,本实验以T7DNA聚合酶对一DXFD52相关人肝细胞cDNA(DE)进行了分段部分测定,并将所测各部分序列分别在EMBL(欧洲分子生物学库)中进行核酸同源性检索,结果在库中没有找到任何具有同源性的人类基因或DNA片段。因此,我们初步认为DE为一新的人类基因cDNA片段。同时为初步探讨DE的功能,我们还成功地将DE构建于反转录病毒载体PLXSN上。 相似文献
10.
S. Armeanu I. Haessler R. Saller M. G. Engelmann F. Heinemann E. Krausz J. Stange S. Mitzner B. Salmons W. H. Gunzburg S. Nikol 《Journal of microencapsulation》2013,30(4):491-506
Long-term benefits of coronary angioplasty remain limited by the treatmentinduced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirusproducing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ betagalactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of ~ 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy. 相似文献