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Objective To research the consistency of testing results with three different antimajor histocompatibility complex class Ⅰ-related chain A(MICA) specific antibody reagents in order to evaluate their clinical application's value.Method An collaborative study of 18 laboratories was undertaken at the 16th International HLA and Irnmunogenetics Workshop.Total of 16 sera(4 batchs)were tested for anti-MICA antibodies by Luminex method with three different reagents (Kit-A,-B and -C).Result Anti-MICA antibodies were found in 15 sera,except one sera(no.S04) ; No.S10 sera showed positive results in all the laboratories.The anti-MICA antibodies were divided into MICA-G1 group (MICA01,02,07,12,17 and 18) and MICA-G2 group (MICA 04,06,08/27,09 and 19).MICA-G1 group specific antibodies were detected in 5 sera with Kit-A and-B reagent; but there were false-positive results of anti-MICA08/27 and MICA19 antibodies in this 5 sera with Kit-C.MICA-G2group specific antibodies can be detected in other 5 sera with Kit-A and-B,But the MICA specific antibodies testing gave different results with Kit-A,-B and-C in all the last 5 sera samples.Testing of MICA08/27 showed highest consistency results (86.67％,13/15) with Kit-A,-B and-C; and testing of MICA19 showed lowest consistency results (40％,6/15) with this 3 reagents.There were 80％ consistency results of anti-MICA specific antibodies in 13 sera with Kit-B.Conclusion There are the same effect to judgment positive or negative result for anti-MICA antibodies with 3 different reagents,but the results of anti-MICA specific antibodies are not the same.Therefore,it's better to use two or more reagents to test anti-MICA specific antibodies,or choose reagent with wide detection range.
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Objective To construct a recombinant adenovirus vector ( Ad-hIL-17F) expressing human interleukin 17F (hIL-17F) and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 (QBI-293A) cells to construct Ad-hIL-17F.RT-PCR analysis and indirect immunofluorescent assay (IFA) were performed to determine the expressions of hIL-17F.MTT assay was used to detect the inhibitory effects on cell growth of ECV 304 .The expressions of VEGF and Ang-1 in 293 A and ECV304 cells were measured by ELISA .The effects of Ad-hIL-17 F on the expressions of VEGF in 293A cells were analyzed by Real-Time PCR.Results The sequencing result verified that hIL-17F gene fragment was correctly inserted in the vector .The expressions of hIL-17F gene at mRNA and pro-tein levels were confirmed by RT-PCR and IFA.Ad-hIL-17F could significantly inhibit the growth of ECV304 cells and down-regulate the expressions of VEGF and Ang-1 in 293A and ECV304 cells.Conclu-sion Ad-hIL17F expressing hIL-17F was successfully constructed .The expressed hIL-17F could inhibit the angiogenesis through down-regulating the expressions of VEGF and Ang-1.
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Objective To establish Ad-LIF-OSM transgenic feeder cells for the expansion of CD34+ hematopoietic stem/progenitor cell and tentatively study its effect in expansion and differentiation of cord blood hematopoietic stem cell(HSC) in vitro.Methods In the foundation of pAdTrack-CMV-LIF-polyA+promoterΔ,the OSM gene was inserted to the vector plasmid.Then we structure the transfer plasmid pAdTrack-CMV-LIF-polyA+promoterΔ-OSM.The transfer vector and backbone vector were further cotransfected for homologous recombination. The result pAdEasy-1-pAdTrack-CMV-LIF-polyA + promoterΔ-OSM homologous recombination plasmid were transfected into the human embryonic kidney 293 (QBI-293A) cells,leading to formation of the recombinant adenviruses Ad-LIF-OSM which co-expressing LIF and OSM.Infect the feeder layer cells with groups of Adenovirus,detection the expressing of LIF and OSM in WI-38 cells by RT-PCR and ELISA.Compares the stem cells differentiation and proliferation of the different experimental groups in vitro by transwell and cell counting.Results The sequencing results show that the OSM genes were anastomotic in Ad-LIF-OSM.LIF and OSM gene could be detected in feeder layer cells which infected by Ad-LIF-OSM.Exogenetic LIF and OSM have special effect in culturing HSC in vitro.Conclusion The adenoviral vector co-expressing LIF and OSM (Ad-LIF-OSM) were successfully constructed.Ad-LIF-OSM transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate.
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Objective To evaluate the effects of combined administration of recombinant human interleukin-11(rhIL-11),recombinant human G-CSF(rhG-CSF)and recombinant human interleukin-2 (rhIL-2)on acute radiation sickness(ARS)beagles.Methods Sixteen beagle were irradiated with 4.5 Gy60 Co γ-rays to establish ARS models,and were divided into irradiation control group,supportive care group and combined cytokines treatment group.After irradiation irradiation control group was given no treatment,the dogs in supportive care group received purely symptomatic treatment,while combined cytokines treatment group received rhIL-11 50μg/(kg·d)and rbG-CSF 10μg/(kg·d)subcutaneously(0-14 d)and rhIL-2 1×1 06 U/d(29-43 d)besides symptomatic treatment.Manifestation and characteristics of ARS beagles were observed,and the survival time were recorded.At last,post-mortem examination and histological examination were performed.Results All animals underwent nausea,diarrhea and fever.After irradiation,all animals in irradiation control group died in two weeks,and the mean survival time was 12.7 d,while only one died at 33 d in supportive care group.All dogs in combined cytokine group survived at 45 day after exposure,and their haematopoiesis and gastrointestinal tract were recovered.Conclusions Combination of rhIL-11 + rhG-CSF + rhIL-2 treatment could be significantly effective on ARS beagles irradiated by 4.5 Gy60 Co γ-rays,which could accelerate injured haemotopoiesis and intestinal tract recovery,increase the survival rate and improve the life quality of animals.
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Objective To explore the mechanisms of cytokines on acute radiation disease in irradiated beagles.Methods The sera of beagles irradiated with 4.5 Gy γ-rays with cytokines treatment was collected at different time points post irradiation.The two-dimensional gel electrophoresis(2-DE)was used to isolate and compare the differentially expressed proteins in sera.HD-MS was used to analyze the differentially expressed proteins with significance,and the amino acid sequences should be determined. Results High resolution 2-DE gel map was obtained.There were six differentially expressed proteins in sera of irradiated beagles at different time points.Four protein spots were successfully identified by MS.A significant spot was identified as serum amyloid A(SAA)by HD-MS,with relative molecular mass of 13 077 and isoelectfie point of 6.26.Expression of SAA was not found 1 d pre-irradiation and 36 d postirradiation,but increased slightly 1 d(0.2166)and significantly 14 d post-irradiation(0.4577). Conclusions The expression of serum amyloid A was consistent with the process of acute radiation injury,which might indicate the turnover of the disease.
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Objective To explore the clotting mechanism in beagles irradiated by 4.5 Gy γ-rays after treatment with supportive care,or supportive care and combined cytokines.Methods Sixteen beagles were divided into irradiation control group,Supportive care group and combined cytokines treatment group.Platelet aggregation test,thrombelagtography (TEG) and the time measurement were analyzed in vitro.Results In irradiation group and supportive care group,the platelet aggregation rates in beagles were decreased markedly and the k value of TEG was increased 7 d post-irradiation,while those indexes in combined cytokines treatment group changed little.At 14 d post-irradiation,each parameter of TEG in irradiated group changed obviously.The values of r,k,r+k and M were elevated significantly,clotting time and the maximum coagulation time of thrombus delayed,the Ma value was decreased markedly,and the maximum elasticity amplitude of thrombus was diminished.All parameters in combined cytokines treatment group were better than those in supportive care group.The thrombin time was prolonged obviously in irradiated group 14 d post-irradiation,while the thrombin time was the longest at 2-3 weeks post irradiation in supportive care group and combined cytokines treatment group(P>0.05).Conclusions Cytokines could improve the platelet aggregation and the blood clotting functions of beagles suffering from acute radiation sickness.
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Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.
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Objective To investigate the mechanism of treatment of granulocyte colony-stimulating factor(rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2 (rhIL-2)on hematopoietic injuries induced by 4.5 Gy60 Coγ-ray irradiation in beagles,and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS).Methods Sixteen beagle dogs were given 4.5 Gy60 Co γ-ray total body irradiation(TBI),then randomly assigned into irradiation control group,supportive care group or cytokines+supportive care (abbreviated as cytokines)group.In addition to supportive care,rhG-CSF,rhlL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group.The percentage of CD34+cells,cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry.Results After 4.5 Gy 60 Co γ-ray irradiation,the CD34+cells in peripheral blood declined obviously(61.3%and 52.1% of baseline for irradiation control and supportive care group separately).The cell proportion of nucleated cells in Go/G1 phase was increased notably(99.27% and 99.49% respectively).The rate of apoptosis(26.93% and 21.29% separately)and necrosis(3.27% and 4.14%,respectively)of nucleated cells were elevated significantly when compared with values before irradiation(P<0.05) 1 d post irradiation.When beagles were treated with cytokines and supportive care,the CD34+cells in peripheral blood were markedly increased(135.6% of baseline).The effect of G0/G1 phase blockage of nucleated cells became more serious(99.71%).The rate of apoptosis(5.66%)and necrosis(1.60%)of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure.Conclusions Cytokines maybe mobilize CD34+cells in bone marrow to peripheral blood,indce cell cycle block at G0/G1 phase and reduce apoptosis,and eventually cure hematopoieticinjuries induced by irradiation.
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Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.
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Objective:To study the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the MG-63 osteosarcoma xenografts in nude mice.Methods:Ad-ING4 was transfected into QBI-293 cells and harvested.15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS,Ad-GFP and Ad-ING4 groups.Then PBS(100 μl),Ad-GFP(100 μl,10~9pfu/ml) and Ad-ING4 (100 μl,10~9pfu/ml) for each one were given respectively QOD for 5 times,with intratumor injections.Tumor volume changes were monitored;and the 15 mice were sacrificed 2 weeks after treatment,the tumors were removed,weighed and ratios of tumor-suppression were calculated.The morphological changes of apoptotic tumor cells were observed under microscope.Bcl-2,Bax,Caspase-3,VEGF,CD34 expression was tested by immumohistochemistry.Results:High titer(10~9pfu/ml)adenoviral vector of ING4 gene were obtained.In nude mice bearing MG-63 osteosarcoma xenografts,the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group.The ratios of tumor weight-suppression of Ad-ING4 group was 59.3%(P＜0.05).Immumohistochemistry displayed that the expression of Bax,Caspase-3 was up-regulated and the expression of Bcl-2,VEGF,CD34 was down-regulated by Ad-ING4.Conclusion:Ad-ING4 can inhibit the growth of MG-63 osteosarcoma xenografts in nude mice,which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.
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Objective:To study the antitumor effect and mechanism of cocultured CIK cells modified with IL-24 gene and autologous DCs on HL-60 cells in vitro.Methods:DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells ( PBMC).IL-24 gene was transferred into CIK cells via electroporation.The cells obtained were named CIK-IL24.RT-PCR and ELISA were used to evaluate expression of IL-24 gene in transfected CIK cells.The phenotypic changes of CIK cells were identified by flowcytometry analysis.The concentration of IFN-γ and TNF-α in supernatant of CIK was determined by ELISA.FCM was used to determine the cytotoxicity of cocultured CIK cells modified with IL-24 gene and autologous DCs against HL-60 cells.Results:Eukaryotic expressing plasmid pcDNA3.0-IL24 was transferred into CIK cells successfully via electroporation.The expressing rate of CD3~+、CD3~+CD56~+ cells had no significant change in CIK cells.However,the rate of CD4~+CD25~+ cells was significantly decreased compared with that of the control group.Expression of adhesion molecules CD54,CXCR4 were significantly increased on CD3+CD56+ cells.CIK-IL24 cells produced markedly higher levels of IFN-γ and TNF-α as compared with the CIK cells.By comparison with non-transfected CIK cells co-cultured with DCs,transfected CIK cells co-cultured with DCs had a significantly higher lytic activity against HL-60 cells.Conclusion:IL-24 gene modification can enhance the anti-tumoral immunity of CIK cells,the mechanism of which might be related to the increased secretion of IFN-γ,TNF-α,up-regulation of adhesion molecule expression,and reduction of the rate of CD4~+CD25~+ cells in CIK cells.
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As a biomaterial to be used for reparation in the case of trauma, the silk fibroin, particularly its effect on the transcription and expression of VEGF gene, is a concern. In this study, the ECV304 cell's growth shape and growth curve on the regenerated silk fibroin film were observed, and its VEGF secretion level was measured by ELISA test. It was found that the regenerated silk fibroin film did not interfere with ECV304 cell's growth and function. The L929 cell transfected with human VEGF gene grew on the regenerated silk fibroin film; the real-time quantitative RT-PCR method and ELISA test were used for detecting the transcription and expression of VEGF gene. The results showed the regenerated silk fibroin film did not interfere with the transcription and expression of VEGF gene. Therefore, the regenerated silk fibroin film is a safe biomaterial for inducing vascularization with no untoward effect on the reparation of trauma.
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Animals , Humans , Biocompatible Materials , Pharmacology , Cell Line , Endothelial Cells , Cell Biology , Metabolism , Fibroins , Pharmacology , Silk , Pharmacology , Transcription, Genetic , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity.
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Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.
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We constructed the recombinant adenovirus vector expressing IL-24 and E1A (Ad-IL-24-E1A) and investigated the inhibition of Ad-IL-24-E1A on SMMC-7721 hepatocellular carcinoma in vitro. We amplified IL-24 gene by PCR using pAdTrack-IL-24 as template. The IL-24 gene was cloned into pAdTrack-IRES at the Bgl II and Sal I site to form pAdTrack-IL-24-IRES. E1A digested from pAdTrack-E1A was cloned into the pAdTrack-IL-24-IRES at the Xho I and EcoR V site to form the pAdTrack-IL-24-IRES-E1A. We co-transformed both pAdTrack-IL-24-IRES-E1A and pAdeasy-1 digested by Pme I and packaged to obtain Ad-IL-24-E1A. Ad-IL-24-E1A at 50 MOI infected SMMC-7721 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay determined cell proliferation. Flow cytometry detected Cell apoptosis. The apoptotic rate of SMMC-7721 cells was 52% 48 h after infection with Ad-IL-24-E1A. The result showed that the growth of SMMC-7721 cells was significantly inhibited by Ad-IL-24-E1A at the MOI of 50.
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Humans , Adenoviridae , Genetics , Metabolism , Adenovirus E1A Proteins , Genetics , Antineoplastic Agents , Pharmacology , Apoptosis , Carcinoma, Hepatocellular , Pathology , Cell Line, Tumor , Cell Proliferation , Genetic Vectors , Genetics , Interleukins , Genetics , Liver Neoplasms , Pathology , Recombinant Proteins , Genetics , PharmacologyABSTRACT
To study the inhibitory effect and anti-cancer mechanisms of interleukin 24 (IL-24) on human osteosarcoma cell MG-63, we delivered IL-24 into MG-63 cells in vitro and in vivo by adenovirus. The expression level of IL-24 was detected by RT-PCR and fluorescence microscope; the growth inhibition, apoptosis rate and apoptosis body were measured by MTT, Flow cytometry and Hoechst staining respectively. Furthermore, we analyzed the expression of bcl-2, bax, caspase3 genes by RT-PCR after overexpression of IL-24. For in vivo study, we first established the MG-63 tumor model by grafting MG-63 cells in athymic nude mice; and then injected Ad-IL-24 into the tumors. Two weeks after injection, we sacrificed the mice, removed the tumors, weighed and calculated the ratios of tumor-suppression. We also detected the expressions of Bcl-2, Bax, Caspase-3 and CD34 with immumohistochemistry. Our in vitro results indicated that Ad-IL-24 was transcribed and translated in MG-63 osteosarcoma cells. More interestingly, IL-24 inhibited the growth of MG-63 cells and induced apoptosis by up-regulation of bax, caspase-3 and down-regulation of bcl-2. The in vivo data showed that IL-24 suppressed the tumor growth conspicuously through down-regulating the expression of bcl-2, and up-regulating the expression of bax, caspase-3. This study would provide evidence for the gene therapy of IL-24 on osteosarcoma.
Subject(s)
Animals , Humans , Mice , Adenoviridae , Genetics , Metabolism , Apoptosis , Genetics , Bone Neoplasms , Pathology , Therapeutics , Caspase 3 , Metabolism , Cell Line, Tumor , Genetic Therapy , Interleukins , Genetics , Mice, Nude , Osteosarcoma , Pathology , Therapeutics , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Recombinant Proteins , Genetics , bcl-2-Associated X Protein , MetabolismABSTRACT
To study the inhibitory effect of a recombinant adenoviral vector carrying human IL-24 gene on SGC-7901 human gastric cancer cell. We infected the SGC-7901 gastric cancer cells with Ad blank adenovirus at various multiplicity of infection (MOIs) to find the optimal infective dose. The SGC-7901 tumor cells were infected with Ad-IL-24 at the optimal MOI in the following experiments. Adenovirus-mediated IL-24 transcription expression in SGC-7901 cells was examined by RT-PCR. The growth-suppressing effect of Ad-IL-24 on SGC-7901 tumor cells was assessed by MTT assay. Apoptosis and cell cycle of SGC-7901 tumor cells infected with Ad-IL-24 was evaluated by flow cytometer (FCM), respectively. The karyomorphology of apoptotic SGC-7901 tumor cells was examined using Hoechst33258 staining under fluorescence microscopy. The expression of apoptosis-related genes was future determined by semi-quantification RT-PCR; We demonstrated that the MOI of 100 was the optimal infective dose in the study on adenovirus-mediated IL-24 gene transfer into SGC-7901 gastric cancer cell; IL-24 gene mediated by adenovirus could successfully transcribe in SGC-7901 tumor cells; Ad-IL-24 could significantly inhibit SGC-7901 tumor cell growth and induce apoptosis, it also can up-regulate the express of bax, caspase-3 and p53 whilst down-regulate the bcl-2 expression. Thus, adenovirus-mediated IL-24 expression had marked anti-tumor effect in suppressing SGC-7901 human gastric cancer cell growth and inducing apoptosis, which may be closely associated with its up-regulation of bax/bcl-2, caspase-3 and p53.
Subject(s)
Humans , Adenoviridae , Genetics , Metabolism , Apoptosis , Genetics , Caspase 3 , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Interleukins , Genetics , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , Recombinant Proteins , Genetics , Stomach Neoplasms , Genetics , Pathology , Transfection , Tumor Suppressor Protein p53 , Genetics , Metabolism , Up-Regulation , bcl-2-Associated X Protein , Genetics , MetabolismABSTRACT
BACKGROUND:Previous studies have demonstrated that regenerated silk fibroin film can promote pcDNA3.0-vascular endothelial growth factor 165(rEGF165) transfected L929 cells to express VEGF.OBJECTIVE:To investigate the effects of regenerated silk fibroin film on expression of cytokines related to angiogenesis in the fibroblasts transfected by adenovirus mediated-VEGF165(Ad-VEGF165).DESIGN,TIME AND SETTING:ontrolled observational cell gene engineering experiment performed by analysis of variance at the Laboratory of Cellular and Molecular Biology,Soochow University between November 2007 and ApriJ 2008.MATERIALS:Regenerated silk fibroin film was provided by Professor Li Ming-zhong,who was from Department of Material Science and Engineering,Soochow University.METHODS:The QBI-293A and WI-38 fibroblasts cultured on the regenerated silk fibroin film.polyvinyl chloride film and (Ad-GFP)and treated by phosphate buffered saline(PBS)for controls.MAlN OUTCOME MEASURES:VEGF mRNA was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR);the expression levels of VEGF,angiogenin 1(Ang 1),fibroblast growth factor 2(FGF2),and platelet-derived growth factor(PDGF)were detected by enzyme-labeled immunosorbent assay(ELISA).RESULTS:The VEGF mRNA expression in the fibroblasts cultured on the regenerated silk fibroin film was increased but that in the fibroblasts cultured on the polyvinyl chloride film was signifcantly decreased(P<0.05).ELISA results demonstrated that not only VEGF gene expression in 293A and WI-38 cells transfected bv Ad-VEGF165 cultured on regenerated silk fibroin film was high,but also Ang 1 expression increased significantly(P<0.05).Meanwhile,the expression levels of FGF2 and PDGF were normalin the fibroblasts cultured on the regenerated silk fibroin film.CONCLUSION:Adenovirus vector can be effciently transfected into fibroblasts cultured on the regenerated silk fibroin film and can express VEGF and Ang 1 protein with highly biological activity,which accelerates angiogenesis.Regenerated silk fibroin film also can maintain the normal expression levels of FGF2 and PDGF,which ale related to wound healing.
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Objective To construct the human IL-24 recombined adenovirus(Ad-IL24)and to observe the effect of Ad-IL24 on ex vivo culture of HL-60 cells.Methods pAdTrack-CMV-IL24 was constructed by PCR with pcDNA3.0-IL24 recombined plasmid as a template,enzyme digestion and ligation.The pAdTrack-CMV-IL24 lineared by Pme Ⅰ was co-transformed into BJ5183 with pAdEasy-1.The pAdEasy-1-pTrack-CMV-IL24 recombined adenovirus vector was lineared with Pac Ⅰ and then transfected in to QBI-293A cells.The Ad-IL24 was obtained and used to infect HL-60 ceils,and the effect on HL-60 cells was tested by LSCM,Mrrr,FCM,ELISA and immunohistochemistry staining.Results The pAdEasy-1-pTrack-CMV-IL24 vector was constructed and the Ad-IL24 was successfully obtained.The effect of inhibiting growth of HL-60 cells and inducing cell apoptosis by IL-24 was proved by LSCM,MTT and FCM.IL-24 down-regulated the expression of anti-apoptosis factor Bcl-2 and increased the secretion of IFN-γ,TNF-α of HL-60 cells.Conclusion Ad-IL24 can inhibit the growth of HL-60 cells and induce cell apoptosis through down-regulating expression of anti-apoptosis factor and increasing the secretion of IFN-γ,TNF-α of HL-60 cells.The human Ad-IL24 may provide a basic study for the future target therapy to tumors.
ABSTRACT
The biological activities i. e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-λ1 and hIFN-ε were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-λ1-His and pcDNA3.1A-hIFN-ε-His by PCR was constructed, then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells ) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFNλ1-His and rhIFN-ε-His, meanwhile MTT assay was used to detect their antineoplastic activities. It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-λ1-His is more powerful than of rhIFN-ε -His. The antiviral molecular mechanisms of both hIFN-λ1 and hIFN-ε are related to MxA. The foundation for further study on the bioactivities and mechanism of action of hIFN-λ1 and hIFN-ε was established.