Biomedical subjects

Guan-Xin Shen

Publications and source records attributed to Guan-Xin Shen.

6 recordsLinked to original sources

PubMed2006

[In vitro anti-tumor effect of CTL induced by HSP70-Id complex-modified dendritic cells].

OBJECTIVE: To prepare the tumor antigen peptide complex (HSP70-1d) of HSP70 and idiotype (Id) from SmIg ScFv fragment in patients with Chronic B cell leukemia (B-CLL), and to study the anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HSP70-Id complex-modified dendritic cell (DC) in vitro and explore their immune mechanism. METHODS: Purified HSP70 was combined into peptide complex (HSP70-Id) with the prepared Id-ScFv from B-CLL cells in vitro by using biochemical technique. The plastic-adherent monocytes from human peripheral blood were cultured and induced into DC with rhGM-CSF and rhIL-4 using cell culture and separation technique. The cultured DC were harvested and pulsed by HSP70-Id complex. DC morphology was observed under converted phase microscope and its phenotype was characterized by FCM on 8th day as well as their secreting cytokines were measured. Host lymphocytes were stimulated by DC loaded with HSP70-Id complex and co-cultured in the medium containing IL-2. The activation and proliferation of lymphocytes were examined by MTr test, which was also used to assay cytotoxicity of CTL elicited by modified DC to Daudi, K562 and HepG2 tumor cells, and FCM analyzed the changes of T lymphocyte subsets. RESULTS: Mature DCs were obtained successfully, showing typical morphology and phenotypic properties, the expression ratio of cellular surface molecules, CD1a was 20% - 30%, CD83 was more than 72% , both CD86 and HLA-DR over-expressed obviously in the complex-loaded DC group secreting cytokines of Thl type, IL-12 and TNF-alpha. The culturing lymphocytes that were activated by modified DC could more effectively and specifically kill Daudi (71. 24%), but not K562 and HepG2 tumor cells. Results of FCM assay demonstrated that percentage of CD4+ and CD8+ T lymphocytes cocultured with complex-modified DC increased notably to 56.51% and 70.21%, respectively. CD4+ T/ CD8+ T proportion was changed from 1.49 to 0.81. The dose of peptide would be reduced to 1/50 if specific CTL induced by complex-modified DC instead of directly by peptide complex. CONCLUSION: DCs modified by HSP70-Id complex exhibit powerful biological activities, and could induce CTL to specific cytotoxicity against carcinoma cells. It might be produced by cooperation of CD4+ T, CD8+ T lymphocytes and DC. The results also suggested that DC modified by HSP70-Id complex can present antigen and induce CTL with high efficacy and specificity.

Cell Line, Tumor↗

PubMed2005

Effects of four regulating-intestine prescriptions on pathology and ultrastructure of colon tissue in rats with ulcerative colitis.

AIM: To observe different histomorphologic changes of ulcerative colitis (UC) rats that were treated with four regulating-intestine prescriptions (FRIP), to investigate the curative effects of FRIP and to analyze their treatment mechanism. METHODS: The UC rat model was made by the method of 2,4-dinitro chloro benzene (DNCB) immunity and acetic acid local enema. Ninety-eight SD rats were randomly divided into seven groups, namely, the normal control group, model group, salicylazosulfapyridine (SASP) group, Wumeiwan (WMW) group, Baitouwengtang (BTWT) group, Senglingbaishusan (SLBSS) group, and Tongxieyaofang (TXYF) group. Each group had 14 rats (with equal ratio of male and female). The six animal model groups of UC -SASP, TXYF, WMW, BTWT, SLBSS, TXYF-were treated by distilled water except the normal control group. Changes of the rat's general conditions after treatment were respectively observed, the colon tissue damage scores were given out, the pathology of colonic mucosa and changes of ultrastructure were analyzed. RESULTS: Different pathological changes on histology were shown after treatment by FRIP. The colon tissue damage score in model group was higher than that of FRIP groups and SASP group (q = 4.59, 4.77, P<0.05 or q = 5.48, 6.25, 5.97, P<0.01). The scores of WMW group, BTWT group and SLBSS group were lower than that of SASP (q = 4.13, P<0.05 or q = 5.31, 5.12, P<0.01). There was no remarkable difference between the damage score of TXYF group and SASP group (q = 3.75, P>0.05). In addition, some apoptosis cells were found in the pathologic control group. CONCLUSION: The model made with DNCB and acetic acid was successful, and FRIP had better curative effect and WMW was the best curative effect, BTW, SLBSS and TXYF were similar to SASP, and we discovered that apoptosis was possibly related to UC.

Animals↗

PubMed2005

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Carcinoma, Hepatocellular↗

PubMed2005

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). METHODS: The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

Alkaline Phosphatase↗

PubMed2005

Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor.

To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy. In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells, which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.

Animals↗

PubMed2002

[Construction of expression vector for recombinant annexin II and characteristics of its fibrinolysis].

The study was designed to investigate annexin II resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexin II vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. Annexin II gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexin II was transfected into HL-60 cells and its distribution in the cell and structure characteristics of annexin II protein were evaluated by multi-photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexin II antisense oligonucleotide (AS) targeting to the start site of annexin II cDNA. The results showed that the recombinant pZeoSV2(+)/ANN II was constructed successfully and expressed in HL-60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occurred higher level of expression. The level of the plasmin was significantly enhanced in the present of annexin II. The FCM and Western blot analysis showed the annexin II expression was similar both in transiently and stably transfected in HL-60 cells. Annexin II antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexin II plays an important role in the fibrinotysis. Annexin II vector was defined as a expression tool for further studying fibrinolysis and coagulopathy in malignant disease.

Annexin A2↗