Biomedical subjects

Yu-quan Wei

Publications and source records attributed to Yu-quan Wei.

29 records · Page 2Linked to original sources

PubMed2004

[Prokaryotic expression, purification and refolding of extracellular ligand binding domains of chick Tie-2 and its immunogenicity].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E. coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis. RESULTS: The recombinant protein was highly expressed in E. coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein. CONCLUSION: The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.

Animals↗

PubMed2004

[Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis. RESULTS: The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density. CONCLUSION: The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.

Angiogenesis Inhibitors↗

PubMed2004

[Constructing the tumor cell vaccine based on homologous matrix metalloproteinase-2 and exploring its effects].

OBJECTIVE: To construct the tumor cell vaccine based on homologous matrix metalloproteinase-2 (MMP-2), and explore its anti-tumor effects. METHODS: The tumor cell vaccine was constructed by transfecting chicken MMP-2 to tumor cells. MTT colorimetric assay was used to evaluate the activation of the transfectants. Then we observed the formative tumor response in vivo and evaluated the effects of the tumour cell vaccine in immunized mice rechallenged with parental cells. RESULTS: Using RT-PCR, we picked out the cell strain with the highest expression level from the transfectants. The MTT colorimetric assay revealed that the transfectants increased the expression of the MMP-2 of tumor cells while they did not change the trend of tumor cells' growth. And in animal experiments the vaccine was preliminarily found to be effective for inhibiting the tumour growth and prolonging the survival time of animals. CONCLUSION: We have constructed the tumor cell vaccine based on homologous matrix metalloproteinase-2. The experiments demonstrated that breaking the immune tolerance of MMP-2 by means of active immunity should be a useful approach to cancer therapy.

Adenocarcinoma↗

PubMed2004

[Serological screening of xenogeneic antigens from rat testis].

OBJECTIVE: It is the intent of this study to analyzed the rat testis library by employing a modified SEREX (serological analysis of recombinant cDNA expression library) approach to find the xenogeneic homologous tumor antigens which could be useful in developing cancer vaccines. METHODS: The screening serum was obtained from the immunized rabbits with human ovarian cancer cells, and 10 positive clones were isolated from the rat testis cDNA library using SEREX technology. RESULTS: We found these 10 clones encoded seven different proteins by means of bioinformatic analysis. Among them, OV-2 and OV-4 encode proteins related to carcinoma in human, yet OV-6 and OV-7 are novel and their encoded proteins remain unknown. CONCLUSION: This study indicates that the utilization of xenogeneic immunized serum in the serological screening of xenogeneic homologous tumor antigens may expand the application of traditional SEREX technology.

Animals↗

PubMed2004

[Construction of pcDNA-BLC and expression of BLC in tumor cells].

OBJECTIVE: To establish a stable transformants of B-lymphocyte chemoattractant (BLC) with the eukaryotic expression vector for investigation of its anti-tumor function further. METHODS: The BLC full-length cDNA was amplified by RT-PCR from the total RNA isolated from the spleen of mouse. After sequence determination, the BLC full-length cDNA was cloned into the eukaryote expression vector pcDNA3.1 (+), the resulted recombinant was designated as pcDNA-BLC. Then pcDNA-BLC was transfected into murine tumor cell line colon 26 by LIPOFECTIN. The stable transformants were selected by G418 and identified by immunoblot assay. The bioactivity of BLC was confirmed by chemotaxis assay. RESULTS: The recombinant pcDNA-BLC was successfully constructed. The stable transformants of BLC can express the bioactive BLC in tumor cell lines. CONCLUSION: The stable transformants can stably express BLC in tumor cell lines. These stable transformants will be useful for further study of the BLC biological effect on tumor and for the development of cancer vaccine.

Adenocarcinoma↗

PubMed2004

[Inhibition of malignant ascites with endostatin adenoviral vector].

OBJECTIVE: To evaluate the therapeutical effect of endostatin adenoviral vector on malignant ascites and compare it with the combined effect of ad-endostatin and DDP. METHODS: We transfected SKOV3 cells with ad-endostatin to inhibit human umbilical vein endothelial cells (HUVEC) proliferation. Then we administered ad-endostatin intraperitoneally in BALB/c mice to detect the serum endostatin expression and in the mice with malignant ascites to evaluate the therapeutic efficacy. RESULTS: Recombinant endostatin inhibited HUVEC proliferation. Endostatin was expressed in serum after intraperitoneal injection of ad-endostatin. The effects of endostatin on inhibition of the ascites accumulation and peritoneal capillary permeability, on demotion of VEGF protein level and microvascular density in tumor tissues, on reduction of the number of red cells and tumor cells counted in malignant ascites, and on prolongation of mice survival were significant in two different malignant ascites. Furthermore, the combination of ad-endostatin with DDP increased the therapeutic efficacy. CONCLUSION: These data suggested that endostatin gene therapy may represent a potential new treatment for malignant ascites, and the therapeutic efficacy can be enhanced by a combination of ad-endostatin with chemotherapy.

Adenoviridae↗

PubMed2004

[Construction of eukaryotic expression plasmid pSecTag2B-msBlyS expressing mouse soluble B lymphocyte stimulator].

OBJECTIVE: The purpose of this study was to clone the soluble form of the mouse BlyS (msBlyS) and insert it into a eukaryotic expression vector pSecTag2B in order to further elucidat the antitumor activity induced by msBlyS expressed by the recombined plasmid pSecTag2B-msBlyS. METHODS: Full length cDNA of mouse soluble BlyS (msBlyS) was amplified by reverse transcription-PCR from total RNA of mouse spleen. The PCR product was ligated directly with linearized vector pCR2.1 supplied in the TA cloning kit. The recombined plasmid pCR2.1-msBlyS which was selected and identified using blue-white screening method and restriction map analysis and the purified original plasmid pSecTag2B were both cut by HindIII and EcoR I. The digested fragments were extracted and purified from low-melting temperature agarose and ligated by T4DNA ligase. The recombined plasmid pSecTag2B-msBlyS were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. RESULTS: The sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation. CONCLUSION: Eukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.

Animals↗

PubMed2004

[The effects of transferring DPC4-adenovirus on pancreatic adenocarcinoma cells].

OBJECTIVE: To observe the effect of transferring DPC4-adenovirus on pancreatic cells. METHODS: The recombined wild DPC4 gene and replication-deficiency adenovirus was transfected into cultured pancreatic adenocarcinoma cells and exograft pancreatic adenocarcinoma at the axillary flank of nude mice. The expression of objective gene was evaluated by flow cytometry and immunohistological chemistry assay. The cell cycle was also measured by flow cytometry. The related data were collected and analyzed with statistical methods. RESULTS: in viro, the suppression rate of transferring DPC4-adenovirus on cultured pancreatic adenocarcinoma cells was 30% on the fourth day. The fluorescence intensity of PD cells, P cells, HD cells, PA (Pc-3-Adenovirus) cells was higher than that of HS766T cells and HA cells (P<0.05). After transfection, PD cells proliferated most slowly, while H and HA proliferated most quickly (P<0.05). Significant difference was seen between the volume of exograft tumor of the controlled group and that of the treatment group (P<0.05). After transfection, the proliferation of cultured pancreatic adenocarcinoma cells was suppressed, the cells of G1 phase increased obviously (P<0.05) while the cells of S phase decreased (P<0.05), although no obvious increase of apoptosic cell number was observed. CONCLUSION: The DPC4-adenovirus can be transfected into pancreatic adenocarcinoma cells effectively. It could suppress the proliferation of pancreatic adenocarcinoma cells in vivo and in vitro.

Adenocarcinoma↗

PubMed2004

[Inhibition of tumor growth and metastasis via local administration of recombinant human endostatin adenovirus].

OBJECTIVE: The growth and metastasis of solid tumors are dependent on angiogenesis. Endostatin, the C-terminal proteolytic fragment of collagen XVIII, is a potent endogenous angiogenesis inhibitor. The authors designed a topical antiangiogenic gene therapy with recombinant human endostatin adenovirus (Ad-hEndo) and assessed its effects on the inhibition of angiogenesis in vitro, and tumor growth and metastasis in vivo. METHODS: Malignant cells (A549) were infected with Ad-hEndo. The expression of recombinant protein and the inhibition of cultured human umbilical vein endothelial were investigated. Immunodeficient A549 nude mice were treated with intratumoral injection of Ad-hEndo, the empty vector Ad-control or saline (NS). The dose-response, side effects, and serum concentration of endostatin were observed. RESULTS: Recombinant endostatin protein was detected in the infected tumor cells with different MOI Ad-hEndo and its inhibitory effect on endothelial cells growth was shown. In animal study, the volume of tumor and the number of pulmonary metastatic lesions in the Ad-hEndo treatment group were significantly smaller than those in the control groups (P<0.05). CONCLUSION: The present findings provide evidence of the anti-tumor effects of the endostatin and may be important for the further use of it in topical antiangiogenic gene therapy of cancer.

Adenoviridae↗

PubMed2003

Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice.

Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.

Alginates↗

PubMed2003

Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor.

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4(+) T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8(+) cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8(+)) or CD8-depleted (CD4(+)) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-gamma and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.

Adoptive Transfer↗