[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vect...[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.展开更多
Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the...Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.展开更多
In recent years,polarization remote sensing has garnered increasing attention,particularly within the realm of meteorology.To accurately simulate polarization information,the vector discrete-ordinate radiative transfe...In recent years,polarization remote sensing has garnered increasing attention,particularly within the realm of meteorology.To accurately simulate polarization information,the vector discrete-ordinate radiative transfer(VDISORT)model developed earlier by the community is further enhanced to an advanced version(referred to as A-VDISORT)through an improved ocean surface reflection.The Fresnel reflection matrix,which includes wind-generated roughness and shading effects,is served by an ocean bidirectional reflection distribution function(BRDF).The simulation from AVDISORT is compared with SCIATRAN for a Rayleigh scattering atmosphere,and the influence of water-leaving radiance is analyzed by the PSTAR(Polarized System for Transfer of Atmospheric Radiation) model.For GaoFen-5 Directional Polarimetric Camera(DPC) observations with polarization and multi-angle information,clear-sky pixel recognition over the ocean is first carried out.The DPC reflectance of clear conditions is normalized and compared with the observations.It is shown that A-VDISORT has a high simulation accuracy with a bias of –0.0053.The difference between simulation and observation exhibits a standard normal probability distribution function.展开更多
The two-body orbital transfer problem from an elliptic parking orbit to an excess veloc-ity vector with the tangent impulse is studied.The direction of the impulse is constrained to be aligned with the velocity vector...The two-body orbital transfer problem from an elliptic parking orbit to an excess veloc-ity vector with the tangent impulse is studied.The direction of the impulse is constrained to be aligned with the velocity vector,then speed changes are enough to nullify the relative velocity.First,if one tangent impulse is used,the transfer orbit is obtained by solving a single-variable function about the true anomaly of the initial orbit.For the initial circular orbit,the closed-form solution is derived.For the initial elliptic orbit,the discontinuous point is solved,then the initial true anomaly is obtained by a numerical iterative approach;moreover,an alternative method is proposed to avoid the singularity.There is only one solution for one-tangent-impulse escape trajectory.Then,based on the one-tangent-impulse solution,the minimum-energy multi-tangent-impulse escape trajectory is obtained by a numerical optimization algorithm,e.g.,the genetic method.Finally,several examples are provided to validate the proposed method.The numerical results show that the minimum-energy multi-tangent-impulse escape trajectory is the same as the one-tangent-impulse trajectory.展开更多
The radiative transfer model (RT3), a vector radiative transfer (VRT) scheme in a plane-parallel atmosphere, was bounded by a rough ocean surface in this study. The boundary problem was solved using a Fourier series d...The radiative transfer model (RT3), a vector radiative transfer (VRT) scheme in a plane-parallel atmosphere, was bounded by a rough ocean surface in this study. The boundary problem was solved using a Fourier series decomposition of the radiation field as a function of the azimuth. For the case of a rough ocean surface, the decomposition was obtained by developing both the Fresnel reflection matrix and the probability distribution of the water facet orientation as Fourier series. The effect of shadowing by ocean surface waves was also considered in the boundary condition. The VRT model can compute the intensity and degree of polarization of the light at the top of the atmosphere (TOA), the ocean surface, and any level of the atmosphere in the ocean-atmosphere system. The results obtained by our model are in good agreement with those computed by Ahmad’s model. The simulated results showed that the shadow effects of wave facets on the intensity and the degree of polarization are negligible except at the ocean surface near the grazing angle, possibly because we did not consider the effect of white caps.展开更多
A new method of multi-coupled single scattering （MCSS） for solving a vector radiative transfer equation is de- veloped and made public on Internet. Recent solutions from Chandrasekhar＇s X-Y method is used to valida...A new method of multi-coupled single scattering （MCSS） for solving a vector radiative transfer equation is de- veloped and made public on Internet. Recent solutions from Chandrasekhar＇s X-Y method is used to validate the MCSS＇s result, which shows high precision. The MCSS method is theoretically simple and clear, so it can be easily and credibly extended to the simulation of aerosol/cloud atmosphere＇s radiative properties, which provides effective support for research into polarized remote sensing.展开更多
In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.H...In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.展开更多
The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells...The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.展开更多
AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by i...AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.展开更多
Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-B...Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.展开更多
Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally pre...Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.展开更多
A cell model to describe and optimize heat and mass transfer in contact heat exchangers for utilization of exhaust gases heat is proposed. The model is based on the theory of Markov chains and allows calculating heat ...A cell model to describe and optimize heat and mass transfer in contact heat exchangers for utilization of exhaust gases heat is proposed. The model is based on the theory of Markov chains and allows calculating heat and mass transfer at local moving force of the processes in each cell. The total process is presented as two parallel chains of cells (one for water flow and one for gas flow). The corresponding cells of the chains can exchange heat and mass, and water and gas can travel along their chains according to their transition ma-trices. The results of numerical experiments showed that the most part of heat transfer occurs due to moisture condensation from gas and the most intense heat transfer goes near the inlet of gas. Experimental validation of the model showed a good correlation between calculated and experimental data for an industrial contact heat exchanger if appropriate empirical equations were used to calculate heat and mass transfer coefficient. It was also shown that there exists the optimum height of heat exchanger that gave the maximum gain in heat energy utilization.展开更多
Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification u...Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.展开更多
BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of ade...BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)展开更多
Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retrovira...Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.展开更多
INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under approp...INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under appropriate conditions,theengrafted liver cells can continue to express liver-specific functions for an indefinite period of time.展开更多
The full-length cDNA encoding human Granulocyte macrophage colony stimulating factor (GM CSF) was cloned by RT PCR, placed under the control of CMV promoter, and inserted into adenovirus vector of E1 substitution...The full-length cDNA encoding human Granulocyte macrophage colony stimulating factor (GM CSF) was cloned by RT PCR, placed under the control of CMV promoter, and inserted into adenovirus vector of E1 substitution type, pAx1cw. Subsequently, the cassette cosmid was cotransfected into 293 cells together with EcoT22I digested Ad5 TPC, and the replication deficient recombinant adenoviruses(Ad) of human GM CSF were generated efficiently by homologous recombination, with the titers of 1.51×10 9pfu/ml. 48 hours after infection with prepared human GM CSF recombinant adenoviruses in vitro, HeLa cells and primary human skin fibroblasts expressed high levels of human GM CSF (80～400ng/ 10 6cells/24hr). These suggest that the recombinant Ad of human GM CSF prepared by COS/TPC method is effective in mediating GM CSF gene transfer and might be used in cancer gene therapy.展开更多
Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus ...Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus produce "killing" effect selectively on the tumor cells exposed to GCV. We constructed a recombinant retroviral vector LHyTK/N by inserting HyTK gene into the retroviral vector LXSN and cutting out the SV4O early promoter. The HyTK gene was transferred into mouse melanoma cell line B16 mediated by a recombinant virus. PCR analysis showed that the HyTK gene was successfully transferred and replication-competent virus was absent. The "killing" effect on B16/HyTK+ cells exposed to GCV (>0. 1 μmol/L) was evident when investigated under light microscope and by live cell counting.展开更多
The purpose to this paper is to study the existence problem of solutions to the vector quasivariational inequality for vector-valued functions inH-space.
The purpose of this study was to evaluate the effect of cytokines on the efficiency of gene transfer into murine hematopoietic progenitors and human K562 cells mediated by retrovirus vectors (RV) containing bacterial ...The purpose of this study was to evaluate the effect of cytokines on the efficiency of gene transfer into murine hematopoietic progenitors and human K562 cells mediated by retrovirus vectors (RV) containing bacterial neomycin-resistant (neoR) gene. The bone marrow cells were preincubated with cytokines and then transfected with supernatant containing retrovirus vectors, each for 24 h. The transfected cells were plated in the semisolid culture with or without G418. The efficiency of gene transfer into hematopoietic progenitors was estimated by biological assay and PCR analysis. The most efficient combination of the cytokines, IL-1α/IL-3/SCF,increased the efficiency of gene transfer into murine CFUGM from 6.04±1. 34% to 43. 60±5. 94%. SCF alone most efficiently facilitated the gene transfer into K562 cells from 19.04±1. 58% to 54.46±2. 13%. The results suggest that the combination of IL-1α/IL-3/SCF can increase efficiency of gene transfer into hematopoietic stem cells (HSC) and progenitors, and in the treatment of acute myeloid leukemia (AML) using autologous bone marrow transplantion (ABMT),SCF can facilitate gene transfer into hematopoietic cells in gene-marking clinical studies.展开更多
文摘[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.
基金Supported by a grant from the National Natural Sciences Foundation of China No 30100189
文摘Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.
基金supported by the National Natural Science Foundation of China(Grant No.U2142212)National Key R&D Program of China(Grant No.2019QZKK,Qinghai Tibet KeKao)the National Natural Science Foundation of China(Grant No.U2242211)。
文摘In recent years,polarization remote sensing has garnered increasing attention,particularly within the realm of meteorology.To accurately simulate polarization information,the vector discrete-ordinate radiative transfer(VDISORT)model developed earlier by the community is further enhanced to an advanced version(referred to as A-VDISORT)through an improved ocean surface reflection.The Fresnel reflection matrix,which includes wind-generated roughness and shading effects,is served by an ocean bidirectional reflection distribution function(BRDF).The simulation from AVDISORT is compared with SCIATRAN for a Rayleigh scattering atmosphere,and the influence of water-leaving radiance is analyzed by the PSTAR(Polarized System for Transfer of Atmospheric Radiation) model.For GaoFen-5 Directional Polarimetric Camera(DPC) observations with polarization and multi-angle information,clear-sky pixel recognition over the ocean is first carried out.The DPC reflectance of clear conditions is normalized and compared with the observations.It is shown that A-VDISORT has a high simulation accuracy with a bias of –0.0053.The difference between simulation and observation exhibits a standard normal probability distribution function.
基金supported in part by the China Postdoctoral Science Foundation funded project(No.2012M520753)the Fundamental Research Funds for the Central Universities(No.HIT.NSRIF.2014307)the Open Fund of National Defense Key Discipline Laboratory of Micro-Spacecraft Technology(No.HIT.KLOF.MST.201303)
文摘The two-body orbital transfer problem from an elliptic parking orbit to an excess veloc-ity vector with the tangent impulse is studied.The direction of the impulse is constrained to be aligned with the velocity vector,then speed changes are enough to nullify the relative velocity.First,if one tangent impulse is used,the transfer orbit is obtained by solving a single-variable function about the true anomaly of the initial orbit.For the initial circular orbit,the closed-form solution is derived.For the initial elliptic orbit,the discontinuous point is solved,then the initial true anomaly is obtained by a numerical iterative approach;moreover,an alternative method is proposed to avoid the singularity.There is only one solution for one-tangent-impulse escape trajectory.Then,based on the one-tangent-impulse solution,the minimum-energy multi-tangent-impulse escape trajectory is obtained by a numerical optimization algorithm,e.g.,the genetic method.Finally,several examples are provided to validate the proposed method.The numerical results show that the minimum-energy multi-tangent-impulse escape trajectory is the same as the one-tangent-impulse trajectory.
基金supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KZCX2-YW-QN201)the National Natural Science Foundation of China (Grant No. 40805010)+2 种基金the National Basic Research Program of China (973 Program, Grant No. 2010CB 950804)Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (Grant No. 2008BAC40B01)supported by a Post-doctoral Fellowship for Space Science and Application
文摘The radiative transfer model (RT3), a vector radiative transfer (VRT) scheme in a plane-parallel atmosphere, was bounded by a rough ocean surface in this study. The boundary problem was solved using a Fourier series decomposition of the radiation field as a function of the azimuth. For the case of a rough ocean surface, the decomposition was obtained by developing both the Fresnel reflection matrix and the probability distribution of the water facet orientation as Fourier series. The effect of shadowing by ocean surface waves was also considered in the boundary condition. The VRT model can compute the intensity and degree of polarization of the light at the top of the atmosphere (TOA), the ocean surface, and any level of the atmosphere in the ocean-atmosphere system. The results obtained by our model are in good agreement with those computed by Ahmad’s model. The simulated results showed that the shadow effects of wave facets on the intensity and the degree of polarization are negligible except at the ocean surface near the grazing angle, possibly because we did not consider the effect of white caps.
基金Project supported by the Science Foundation of the Airborne Remote Sensing System,Large Research Infrastructure of the Chinese Academy of Sciences
文摘A new method of multi-coupled single scattering （MCSS） for solving a vector radiative transfer equation is de- veloped and made public on Internet. Recent solutions from Chandrasekhar＇s X-Y method is used to validate the MCSS＇s result, which shows high precision. The MCSS method is theoretically simple and clear, so it can be easily and credibly extended to the simulation of aerosol/cloud atmosphere＇s radiative properties, which provides effective support for research into polarized remote sensing.
文摘In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.
文摘The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.
基金Supported by the National Natural Science Foundation of China, No. 30571759Social Development Foundation of Shanghai, No. 200253
文摘AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.
基金This work was supported by the National Natural Science Foundation of China(No.30400163).
文摘Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.
文摘Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.
文摘A cell model to describe and optimize heat and mass transfer in contact heat exchangers for utilization of exhaust gases heat is proposed. The model is based on the theory of Markov chains and allows calculating heat and mass transfer at local moving force of the processes in each cell. The total process is presented as two parallel chains of cells (one for water flow and one for gas flow). The corresponding cells of the chains can exchange heat and mass, and water and gas can travel along their chains according to their transition ma-trices. The results of numerical experiments showed that the most part of heat transfer occurs due to moisture condensation from gas and the most intense heat transfer goes near the inlet of gas. Experimental validation of the model showed a good correlation between calculated and experimental data for an industrial contact heat exchanger if appropriate empirical equations were used to calculate heat and mass transfer coefficient. It was also shown that there exists the optimum height of heat exchanger that gave the maximum gain in heat energy utilization.
文摘Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.
文摘BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)
文摘Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.
基金the grant of National Natural Science Foundation of China,No.39600129
文摘INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under appropriate conditions,theengrafted liver cells can continue to express liver-specific functions for an indefinite period of time.
文摘The full-length cDNA encoding human Granulocyte macrophage colony stimulating factor (GM CSF) was cloned by RT PCR, placed under the control of CMV promoter, and inserted into adenovirus vector of E1 substitution type, pAx1cw. Subsequently, the cassette cosmid was cotransfected into 293 cells together with EcoT22I digested Ad5 TPC, and the replication deficient recombinant adenoviruses(Ad) of human GM CSF were generated efficiently by homologous recombination, with the titers of 1.51×10 9pfu/ml. 48 hours after infection with prepared human GM CSF recombinant adenoviruses in vitro, HeLa cells and primary human skin fibroblasts expressed high levels of human GM CSF (80～400ng/ 10 6cells/24hr). These suggest that the recombinant Ad of human GM CSF prepared by COS/TPC method is effective in mediating GM CSF gene transfer and might be used in cancer gene therapy.
文摘Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus produce "killing" effect selectively on the tumor cells exposed to GCV. We constructed a recombinant retroviral vector LHyTK/N by inserting HyTK gene into the retroviral vector LXSN and cutting out the SV4O early promoter. The HyTK gene was transferred into mouse melanoma cell line B16 mediated by a recombinant virus. PCR analysis showed that the HyTK gene was successfully transferred and replication-competent virus was absent. The "killing" effect on B16/HyTK+ cells exposed to GCV (>0. 1 μmol/L) was evident when investigated under light microscope and by live cell counting.
基金Supported by the Science Research Foundation of Xianning Teacher's College( No.K9911)
文摘The purpose to this paper is to study the existence problem of solutions to the vector quasivariational inequality for vector-valued functions inH-space.
文摘The purpose of this study was to evaluate the effect of cytokines on the efficiency of gene transfer into murine hematopoietic progenitors and human K562 cells mediated by retrovirus vectors (RV) containing bacterial neomycin-resistant (neoR) gene. The bone marrow cells were preincubated with cytokines and then transfected with supernatant containing retrovirus vectors, each for 24 h. The transfected cells were plated in the semisolid culture with or without G418. The efficiency of gene transfer into hematopoietic progenitors was estimated by biological assay and PCR analysis. The most efficient combination of the cytokines, IL-1α/IL-3/SCF,increased the efficiency of gene transfer into murine CFUGM from 6.04±1. 34% to 43. 60±5. 94%. SCF alone most efficiently facilitated the gene transfer into K562 cells from 19.04±1. 58% to 54.46±2. 13%. The results suggest that the combination of IL-1α/IL-3/SCF can increase efficiency of gene transfer into hematopoietic stem cells (HSC) and progenitors, and in the treatment of acute myeloid leukemia (AML) using autologous bone marrow transplantion (ABMT),SCF can facilitate gene transfer into hematopoietic cells in gene-marking clinical studies.
