Objective To study the effect of the ECV-304 cells modified with LIF gene on the ex vivo culture of HSC/HPC in cord blood.Methods The ECV-304 cells were infected by Eukaryotic Expression plasmid pcDNA3.0LIF,and the positive ECV-304 cells were obtained by selected with G418.These cells were used to co-culture with CD34+ cells of cord blood.The phenotype of CD34+ and CD34+ CD54+、CD34+ CD62L+ primitive progenitors was detected by flow cytometry.Results The LIF gene can express in ECV-304 cells steadily.ECV-304 cells modified with LIF gene can improve expansion of CB CD34+、CD34+CD54+ and CD34+ CD62L+ cells while sustaining the expression of homing-related adhesion molecule.Conclusion The ECV-304 cells modified with LIF gene can not only significantly expand CB hematopoietic progenitor cells ex vivo,but the expanded CD34+ cells may well retain their homing ability.

Objective:To detect the biological activity of the expression products of the human ?-NGF cDNA in CHO cell line and purified the seperated protein.Methods:Human ?-NGF was transfected with lipofectamine reagent into CHO cell line.The biological activity was analyzed by objection with microscope and the method of MTT.The pure protein was proved by SDS-PAGE analysis.Results:The protein in the culture supernatant of the positive CHO cells transfected with ?-NGF gene could stimulate the growth of PC12 cell line and go into BBB.Conclusion:The target gene expressed successfully in the transfected CHO cell line and had good biological activity. [

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.

Objective:To construct a recombinant prokaryotic expression vector pGEX-5X-3/hIL-17F and express it in E.coli and to explore the biological activities of human IL-17F fusion protein.Methods:The coding sequence of the mature human IL-17F(minus the signal peptide) was amplified from pUCm-T/hIL-17F by PCR and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase(GST) fusion protein. The fusion protein was induced in E.coli BL21 by IPTG and purified by standard methods reported in prokaryotic system. The purified GST-hIL-17F fusion protein was identified by Western blot. The proliferation of ECV304 cells was observed by incubating them with soluble GST-hIL-17F fusion protein by MTT assay. The concentrations of IL-6, IFN-? and TNF-? in the supernatants of ECV304 cells were determined by ELISA. The effect of GST-hIL-17F on the angiogenesis of the chick chorioallantoic membrane was assessed by CAM assay.Results:A 41 kD fusion protein was effciently induced in E.coli BL21 by IPTG, accounting for about 55% of the total bacterial protein. The purified GST-hIL-17F fusion protein was identified by Western blot. GST-hIL-17F fusion protein had obvious biological activity to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion. GST-hIL-17F had a marked antiangiogenic activity.Conclusion:The preliminary study of hIL-17F recombinant prokaryotic expression and its antiangiogenic effect has been successful, which lays a foundation for future research on the mechanism of antiangiogenesis and clinical application of recombinant hIL-17F protein.

Objective:To construct the cells which can express LIF protein steadily and study the effect of rhLIF and IL-24 on HL-60 cells.Methods:ECV-304 cells were infected by eukaryotic expression plasmid pcDNA3-LIF,then the positive cells were selected by G418.The positive cells were collected and their culture supernatant was stored for further use.Expression of LIF mRNA was detected by RT-PCR.The pAdeasy-1-pAdTrack-CMV-IL-24 was extracted from DH5a.The recombined adenovirus vector was lineared with PacI and transfected into 293 cells.The IL-24 recombined adenovirus(Ad-IL-24) was obtained and used to infect HL-60 cells.At the same time,the culture supernatant containing rhLIF was added into the HL-60 cells which was identified as positive expression of IL-24 by RT-PCR.Synergistic effect of the cytokines on HL-60 cells was tested by LSCM, FCM and immunohistochemistry stain assay.Results:The cells expressing LIF protein steadily were constructed successfully, the high titer of the recombined adenovirus(Ad-IL-24) was obtained. Expression of IL-24 in infected HL-60 cells was identified by RT-PCR.The apoptosis of HL-60 cells induced by rhLIF and IL-24 was proved by LSCM ,FCM and immunohistochemistry stain assay. They had synergistic effect.Conclusion:rhLIF and IL-24 can inhibit growth of HL-60 cells and induce apoptosis of the cells.They have synergistic effect.

Aim To construct the eukaryotic expression vector of hIL-24 cDNA,and express it in CHO cells and detect its anti-tumor effect of recombinant hIL-24 protein.Methods Constructed pcDNA3-hIL-24 was identified by endonucleases digestion & PCR.The recombinant expression plasmids were transfected into CHO cells,human hIL-24 expressed in CHO cells was detected with RT-PCR.The apoptosis-inducing activities of recombinant protein hIL-24 was tested by MTT assay,Hoechst& FCM assay,and the expression of IL-6 and IFN-? from PBMC induced by rhIL-24 was tested by ELISA.Results The eukaryotic expression vector pcDNA3-hIL-24 was constructed correctly.Stable expression of human IL-24 in CHO cells was identified with RT-PCR.The apoptosis of A549 cells induced by hIL-24 was proved by Hoechst & FCM assay,and the expression of IL-6 and IFN-? from PBMC induced by rhIL-24 was identified with ELISA.Conclusion The successful stable expression & experimental study of apoptosis effect of human IL-24 gene lay the foundation for the further study of molecular mechanism of hIL-24 on anti-tumors and potential application.

Aim To study the expression of β-NGF gene in the mammlian cells and the biological activity of its expression products. Methods The β-nerve growth factor (β-NGF) cDNA which obtained from the plasmid pGEM-β-NGF by enzyme digestion analysis was cloned into the expression vector pcDNA3 to construct the recombinant plasmid pcDNA3-β-NGF. COS-7-cell line grown to log phase was transfected using lipofectamine reagent. The expression level of β-NGF mRNA and the biological activity were analyzed by Northern blot and observation of neurite outgrowth of PC12 cell line stimulated by supernatant, respectively. Results The β-NGF gene was expressed successfully in COS-7 cell lines. The culture supernatant of the positive COS-7 cells transfected with pcDNA3-β-NGF could stimulate the neurite outgrowth of PC12 cell line. Conclusion The target gene expressed successfully in the transfected COS-7 cell line and had good biological activity.

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.

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.

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.