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Objective To evaluate the expression of G250 mRNA in patients with renal cell carcinoma (RCC) and its clinical significance. Methods A semi-quantitative reverse transcription (RT)-PCR was developed to assay the levels of G250 mRNA obtained from 7 normal renal tissues and 33 cases of RCC.Of the 33 RCC cases,28 had clear cell carcinoma and 5 had non-clear cell carcinoma.By UICC staging, 8 cases were diagnosed with T_1 stage carcinoma,13 with T_2 and 12 with T_3.Pathological grading showed 11 cases of G_1,15 of G_2,and 7 of G_3. Results Among the 33 cases of RCC,G250 mRNA expression was present in 28 cases (84.8%).However, it was absent in all the 7 normal renal tissues.There was a significant difference between the 2 groups (P0.05);while they were 0.48?0.32,0.23?0.16 and 0.11?0.11,respectively,in RCC cases of G_1,G_2 and G_3,which showed that the expression of G250 mRNA was inversely correlated with tumor grades(P
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Objective To study human prostate cancer tissue specific hytk gene therapy vector using prostate specific antigen (PSA) promoter,and to determine its feasibility. Methods A prostate tissue specific hygromycin phosphotransferase thymidine kinase fusion gene(hytk)vector was established with the substitution of CMV promotor in tgCMV/hytk vector with PSA promotor.The vector was transferred into prostate carcinoma cell lines with FuGENE TM 6.Antitumor effects were observed after GCV treatment. Results PSA promoter that actively drives hytk gene was expressed only in the PSA producing prostate cell line(LNCaP).In vitro experiments demonstrated the LNCaP cells transferred by HyTK gene were killed in the presence of GCV treatment. Conclusions Hytk based vector harboring PSA promoter is a novel ideal candidate vector for prostate carcinoma gene therapy.
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Objective To construct radiation inducible-specific hytk gene therapy vector using radiation inducible gene Egr-1 promoter,and to determine its feasibility in bladder carcinoma gene therapy. Methods A radiation inducible-specific hygromycin phosphotransferase-thymidine kinase fusion gene(hytk)vector was constructed by subcloning hytk gene into pCIneo/Egr-1 vector, which was carrying Egr-1 promoter.Then the vector was transferred into bladder carcinoma cells lines EJ with FuGENE TM 6.Antitumor effects were observed after irradiation with 60 Co-rays as well as gancyclovir(GCV) treatment. Results The new cell line,EJ/Egr-1-hytk,incorporated and expressed the hytk gene,which was proved by RT-PCR and Western blotting.Egr-1 promoter actively drived hytk gene expression in the presence of ?-rays in the EJ cells, while lower hytk expression was detected without ?-rays.Antitumor effects were observed after ?-rays irradiation with 60 Co rays and GCV treatment. Conclusions Hytk-based vector harboring Egr-1 promoter is a novel ideal candidate vector for bladder carcinoma gene therapy.
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Objective:To construct DNA vaccines expressing prostate-specific membrane antigen(PSMA) and/or granulocyte-macrophage colony-stimulating factor(GM-CSF) and to determine their immunoactivity.Methods: Recombinant plasmids pIRES-PSMA-mGM-CSF,pIRES-PSMA,and pIRES-mGM-CSF were constructed with DNA vaccine vector pIRES.After identified by endonuclease digestion,the above 3 plasmids and blank pIRES vector were used to immunize C56BL/6 mice(n=15).LDH release assay was used to exam the cytotoxicity of cytolytic T lymphocytes in each group.Results: We successfully constructed the above mentioned recombinant plasmids.Mice in pIRES-PSMA-mGM-CSF immunized group had the highest specific cytotoxicity,followed by pIRES-PSMA and pIRES-mGM-CSF immunized groups.The blank pIRES group had the lowest cytotoxicity(P
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AIM: To investigate the effects of dexamethasone(DEX) on the pancreatic endocrine function when steroid diabetes mellitus(SDM) occurred. METHODS: 140 rats were randomly assigned to 4 groups according to the clinic doses of DEX administration, Radioimmunoassay and in situ hybridization were applied to evaluate islet hormone changes in pancreas and blood.RESULTS: Changes in fasting blood glucose(FBG) ?fasting serum insulin(FINS) caused by DEX were dose and time-dependent. Changes in somatostatin(SS)mRNA and protein in islet as well as FINS occurred earlier than that of FBG. CONCLUSION: The super-physiological DEX influenced the expression SS and insulin secretion in islet, which may play an important role in SDM.
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AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), which could coordinate expression of two genes in a single vector, was optioned. By using liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfp gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy.
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Objective Evaluated NOS expression in bladder tissue from the patients with transitional cell carcinoma (TCC) of the bladder and studied its relationship with angiogenesis. Methods Bladder carcinoma tissue specimens were procured from 58 patients with TCC and 14 cases of benign bladder tissue as contrast group. NOS immunohistochemistry was performed on all tissue specimens and microvessal density (MVD) was counted by endothelial cells immunostained. Results Inducible NOS specific proteins were found in 47 of 58 bladder cancer specimens but not in control bladder tissue. The malignant cells and inflammatory cells within the carcinomas were highly iNOS positive whereas specimens of bladder mucosa outside the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells in both normal urothlium and tumor tissue showed a highly positive eNOS immunostaining but its immunoreaction was not detected in either malignant or benign epithelium. MVD was (39.3?19.5)/HP and (29.3?10.5)/HP in iNOS positive and negative tissues respectively (P