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戏说基因:基因家族中的特殊成员(二)奇云四、争论中的“犯罪基因”马克思早就断言:犯罪必将贯穿于阶级社会发展的全部历史。人为什么会犯罪?是什么驱使行为人疯狂地破坏社会、侵扰他人呢?这个沉重的问题,数千年来一直为无数哲人所冥思苦想。亚里斯多德曾将贫穷、情... 相似文献
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本文在前期工作的基础上,构建了汉滩病毒76-118株M基因G2片段与S基因5'端0.7Kb片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7及pcDNA3.1-S0.7G2;用该质粒免疫BALB/c小鼠,结果表明两种质粒免疫小鼠可同时诱导产生抗滩滩病核蛋白(NP)及糖蛋白(GP)特异性的抗体,且前者刺激产生的抗体效价明显高于后者。淋巴细胞增殖实验表明,pcDNA3.1-G2S0.7组免疫小鼠脾细胞时NP及GP的增殖指数均明显高于空载体对照组,而pcDNA3.1-S0.7G2组未检测到其淋巴细胞有明显的增殖。这说明汉滩病毒M基因G2片段及S基因0.7Kb片段的嵌合基因既可刺激机体产生特异的抗汉滩病毒体液免疫应答,也可刺激机体产生特异的细胞免疫应答。不同拼接方式对嵌合基因免疫效果有很大影响,嵌合基因G2S0.7这种拼接方式明显优于S0.7G2。 相似文献
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果实成熟过程相关调控基因研究进展 总被引:10,自引:2,他引:8
果实成熟过程中,多聚半乳糖醛酸酶(PG)参与果胶的分解,从而在果实软化中起作用,新近发现,果实软化过程中,协同展蛋白具有一定的作用:ACC合成酶(ACS)、ACC氧化酶(ACO)和ACC脱氨酶与乙烯合成直接有关,ACS是乙烯形成的关键酶,由多基因家族编码,各个基因协同表达,每一基因都有自己的转录特性,新近不断发现果实中ACS基因家族中的新成员;ACO是一种与膜结合的酶,这种酶具有结构上的立体专一性 相似文献
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近年来,幽门螺杆菌的分子生物学研究取得了很大进展,而既是定植因子、又是毒力因子的幽门螺杆菌尿素酶也得到了更加深入的研究。本文就近年来幽门螺杆菌尿素酶基因的结构、转录、表达调控等方面一些新的研究进展作一综述。 相似文献
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逆病毒载体介导双耐药基因在脐血CD34^+细胞中的表达和抗性研究 总被引:1,自引:0,他引:1
To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial. 相似文献
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探讨人核糖核酸抑制因子 (hRI)基因在人脐血干细胞中的转染及表达情况 ,及转染后对小鼠B16黑色素瘤生长的影响。用免疫磁珠分离系统 (MACS)分离纯化人脐血CD34+ 细胞后 ,用制备的含hRI基因的逆转录病毒上清转染脐血CD34+ 细胞 ,采用克隆形成法和PCR法检测转染效率 ,Western blot和免疫荧光法检测基因表达 ,同时观察RI对荷瘤C57BL小鼠B16黑色素瘤生长的影响。应用MACS能高度纯化人脐血CD34+ 细胞 ,使分选后的脐血CD34+ 细胞纯度平均达96.15%。hRI基因能够转染到脐血CD34+ 细胞上 ,转染效率达 35% ,Western blot和免疫荧光检测转染后CD34+ 细胞hRI基因有阳性表达。经转hRICD34+ 细胞治疗 ,使小鼠B16黑色素瘤的生长速度减慢 ,成瘤率和瘤重降低 ,成瘤潜伏期延长。 相似文献
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Methylation damage response in hematopoietic progenitor cells 总被引:1,自引:0,他引:1
The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells. 相似文献
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LEI P HE Y SHI W PENG J WU S ZHU H CHEN J SHEN G 《Acta biochimica et biophysica Sinica》2007,39(8):599-607
Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l (WEElHu) and proliferation-stimulating stem cell factor (SCF) was generated. In this study, we selected human umbilical cord blood CD34^+ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^+ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^+ cells from chemotherapyinduced myelosuppression. 相似文献
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Human immunodeficiency virus pseudotypes with expanded cellular and species tropism. 总被引:17,自引:17,他引:0
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D H Spector E Wade D A Wright V Koval C Clark D Jaquish S A Spector 《Journal of virology》1990,64(5):2298-2308
One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells. 相似文献
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Shi PA De Angioletti M Donahue RE Notaro R Luzzatto L Dunbar CE 《The journal of gene medicine》2004,6(4):367-373
BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use. 相似文献
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Mayumi Iwata Hiroyuki Nunoi Ichiro Matsuda Shiro Kanegasaki Takashi Tsuruo Y. Sugimoto 《Human genetics》1998,103(4):419-423
Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide.
One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47
phox
, a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus
vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47
phox
. The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce
Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47
phox
-deficient CGD patients, as an in vitro model of gene therapy for p47
phox
-deficient CGD. The transduced cells expressed both P-glycoprotein and p47
phox
protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production
in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is
possible to achieve 100% correction of the CGD defect in p47
phox
-deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo,
in the gene therapy of a number of inherited diseases.
Received: 8 June 1998 / Accepted: 5 August 1998 相似文献
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Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296
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MacNeill EC Hanenberg H Pollok KE van der Loo JC Bierhuizen MF Wagemaker G Williams DA 《Journal of virology》1999,73(5):3960-3967
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols. 相似文献
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Cells positive for the cell surface marker CD34 from bone marrow or umbilical cord blood form a subset of quiescent, hematopoetic precursors that can establish human hematopoesis in immunodeficient mice and can progress down various differentiation pathways in vitro. They provide a valuable model system in which progression from quiescent to cycling to differentiated states can be linked to changes in chromatin and histone modification. We have used the deacetylase inhibitor sodium butyrate to show that turnover of histone H4 acetates is rapid and comparable in quiescent and cycling CD34+ cells from human umbilical cord blood (CD34+ UBC). Surprisingly, the widely used inhibitor trichostatin A (TSA) had little (cycling cells) or no (quiescent cells) effect on H4 acetylation in CD34+ UBC. Among five cell types examined, CD34+ UBC were unique in expressing all (putative) deacetylases tested (HDAC1, -2, -3, -4, -6, -7, and -8 and SIRT1-4), but no single deacetylase correlated with their TSA resistance. Also, HDAC1, -2, -3, and -6 complexes isolated from CD34+ UBC by immunoprecipitation were all inhibited by TSA in vitro. Thus, TSA resistance of CD34+ UBC is not due to acquired or intrinsic TSA resistance of their deacetylases and may reflect an enhanced ability to process the drug. 相似文献