Synergistic tumor suppression by Ad. RGD-iNG4 in human nasopharyngeal carcinoma cell CNE and its mechanism / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the effect of adenovirus-mediated ING4 with RGD on proliferation, apoptosis and cell cycle of human nasopharyngeal carcinoma cell CNE and explore its probable mechanism.@*METHOD@#CNE cells were infected with Ad. RGD-ING4 and adenovirus vector, ING4 gene expression level was detected by RT-PCR and the target protein expression was tested by Western blot. MTT assay was adopted to evaluate the efect of ING4 on cell growth of CNE, Annexin -V-PE/7-AAD Double staining was used to measure the efect of ING4 on apoptosis, and PI staining was used to measure the efect of ING4 on the cell cycle. Differential expression of P21, Bcl-2 and Bax gene was detected by RT-PCR,and Differential expression of Survivin and Caspase 3 protein was detected by Western blot.@*RESULT@#CNE cells were cultured with Ad. RGD-ING4 for 72 h ,the results showed that ING4 was overexpressed in CNE cells ,the growth of CNE cells was obviously inhibited , apoptosis rate was significantly increased and G2/M phase was arrested apparently. The results of RT-PCR showed that Ad. RGD-ING4 significantly down-regulated the Bcl-2 and up-regulates the Bax and P21 expression in CNE cells, and the difference was statistically significant(P < 0.01). Western blot showed that the expression of Survivin was decreased and Cleaved-Caspase 3 was increased.@*CONCLUSION@#Ad. RGD-ING4 can play the role of tumor suppressor synergies on nasopharyngeal carcinoma cell CNE by down-regulating Bcl-2, Survivin expression and up-regulating P21, Bax and Cleaved-Caspase 3 expression.

Construction of recombinant adenovirus vector expressing hIL-17F and functional study of expressed IL-17 F / 中华微生物学和免疫学杂志

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.

Effect of adenovirus-mediated ING4 and IL-24 co-expression on chemosensitivity to human lung adenocarcinoma in vitro and in vivo / 生物工程学报

To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down-regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.

The effect of adenovirus mediated LIF and OSM co-expressing on proliferation and differentiation of umbilical cord blood CD34+ hematopoietic stem/progenitor cell / 中华微生物学和免疫学杂志

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.

Effects of EPCs transplantation on chronic deep vein thrombosis in rats / 中华普通外科杂志

Objective To study the effect of endothelial progenitor cells(EPCs) transplantation on chronic deep venous thrombosis.Methods Bone marrow-derived mouonuclear cells (BMMNCs) were isolated from rat bone marrow by ficoll and cultured with EGM-2MV medium.A rat model of chronic deep vein thrombosis was established by partial ligation of the inferior vena cava and intravenous injection of thrombosin.Model rats were randomly divided into three groups:A(n =25),EPCs group,1 ml 10~6 EPCs transplantation;B(n = 25),EGM-2MV medium group,1 ml EGM-2MV medium transplantation;C (n =25),control group,without any treatment.After transplantation,HE staining and immunohistochemical staining was conducted to detect recanalization of the inferior vena cava.Western blotting of inferior vena cava thrombosis was used to detect VEGF,bFGF protein expression changes.SPSS13.0 software was used for analysis.Results Compared with group B and C,VEGF,bFGF protein significantly increased in group A.The recanalization capillary density was significantly higher in group A than that in group B,and C (P ＜0.05).The neovascularization was identified by immunohistochemical staining using vWF antibody,as endothelial cells.Conclusions EPCs were the precursor of endothelial cells,when transplanted into the deep vein thrombos,initiating angiogenesis and accelerating organization and recanalization of vein thrombus.

The effect of adenovirus-mediated ING4 expression on breast cancer cells / 中华普通外科杂志

Objective To investigate the inhibitory effect of inhibitor 4 of growth(ING4)delivered by adenovirus on human breast carcinoma cell MDA-MB-231.Methods MDA-MB-231 human breast carcinoma cells were irfected with Ad-ING4.The expression level of ING4 gene was detected by RT-PCR and Western blot;The growth inhibition,cell-cycle alteration,and apoptosis were detected by MTT,Flowcytometry and Hochest33258 staining.respectively.RT-PCR was used to detect the transcription of Bax,Bc1-2,Survivin genes;The expression level of Ang-1 gene was detected by ELISA;Ad-ING4 was given intratumorally in athymic nude mice beating MDA-MB.231 tumors.and then tumor growth was monitored;The expression of Bc1-2,Bax and Caspase-3 was analyzed by immumohistochemistry.Results ING4 was successfully transcribed and translated iU MDA-MB-231 cells:Ad-ING4 significantly inhibited the proliferation and induced G_2/M phase arrest to(24.86±1.24)% and cell apoptosis of MDAMB-231.Intratumoral injection of Ad-ING4 suppressed the tumor growth obviously with a inhibitory rate of 49%;Immumohistochemistry showed that the expression of Bax,Caspase-3 were up-regulated and the expression of Bc1-2.Survivin,CD34 were down-regulated by Ad-ING4.Conclusions Ad-ING4 can inhibit the growth of MDA-MB-231 cells and induce apoptosis in vitro and in vivo.

The anti-tumor effect by adenovirus-mediated ING4 and IL-24 co-expression on hepatocellular carcinoma in vitro / 中华微生物学和免疫学杂志

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.

The effect of endothelial progenitor cells transplantation on microenvironments in a murine model of chronic vein thrombosis / 中华普通外科杂志

Objective To investigate the effect of bone marrow derived endothelial progenitor cells (EPC)transplantation on microenvironments in a murine model of chronic vein thrombosis.EPCs transplantation was evaluated whether it can up-regulate thrombus organization and recanalization associated cytokines(VEGF,ANG-1 and MCP-1). Method EPCs from immature Wister rats' bone marrow were isolated using a Ficoll density gradient centrifugation,and cultured in fibronectin-coated plate in EGM-2M Vmedium.EPCs were harvested on the 10th day,then were transplanted into chronic inferior vens cava thrombus of adult Wister rat through the femoral vein.Rats were divided into three groups:blank control group(group A,sham operation),the control group(group B,the medium injected)and the experimental group(group C,EPCs injected).The rats were sacrificed after 28 days.VEGF,ANG-1 and MCP-1 mRNA was measured by real-time quantitative PCR and protein expression change by Western blotting from IVC and thrombus tissue. Results EPCs were identificated successfullv by immunohistochemistry,immunofluorescence and function,then were transplanted into chronic inferior vena cava thrombus of adult rats.After EPCs transplantation,the VEGF,ANG-1 and MCP-1 mRNA expression in group C expression was significantly up-regulated with statistical significance(P＜0.01)compared with group A and group B in IVC and thrombus tissue by real-time PCR.There was no significant difference between group A and group B (P＞0.05).VEGF,ANG-1 and MCP-1 protein expression were similar to mRNA expression.There was significant increase in group C compared to group A and group B(P＜0.01)and no statistical significance between group A and group B(P＞0.05).Conclusion EPCs deriving from bone marrow may change the microenvimnment of chronic vein thrombus through up-regulating thrombus organization and recanalization associated cytokines(VEGF,ANG-1 and MCP-1).

Effect of feeder cells transduced with Ad-hLIF on the expansion of CD34+ cells in indirect co-culture / 中华微生物学和免疫学杂志

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.

Adenovirus mediated IL-24 gene expression inhibits growth of human glioma cell in vitro / 生物工程学报

To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1alpha, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis-related genes.

Construction of adenovirus vector expressing IL-24 and E1A and its inhibition of SMMC-7721 / 生物工程学报

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.

Interleukin 24 inhibits growth and induces apoptosis of osteosarcoma cells MG-63 in vitro and in vivo / 生物工程学报

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.

Adenovirus mediated IL-24 gene expression suppresses gastric cancer cell growth in vitro / 生物工程学报

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.

Effect of regenerated silk fibroin film on transcription and expression of vascular endothelial growth factor gene / 生物医学工程学杂志

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.

The growth-inhibition of osteosarcoma xenografts by adenovirus-mediated ING4 gene in nude mice / 中国免疫学杂志

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.

The cytotoxicity of IL-24 gene-modified CIK cells to HL-60 cells / 中国免疫学杂志

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.

Enhancing effect of IL-24 gene modified dendritic cells co-cultured with CIK cells on cytotoxicity against A549 cells / 中华微生物学和免疫学杂志

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.

Effect of Ad-IL24 on the ex vivo culture of HL-60 cells / 中华微生物学和免疫学杂志

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.

Effects of regenerated silk fibroi film on cytokine expression in the Ad-VEGF165 transgenic fibroblast / 中国组织工程研究

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.

Expression of Human Interferon-λ1 and Interferon-ε Gene in WI-38 Cells and Comparison of Their Biological Activity / 中国生物工程杂志

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.