Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Influence of hepatic arterial blockage on blood perfusion and VEGF,MMP-1 expression of implanted lver cancer in rats

AIM: To investigate the influence of hepatic arterial blockage on blood perfusion of transplanted cancer in rat liver and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1), and to explore the mechanisms involved in transarterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODS: Walker 256 carcinosarcoma was transplanted into rat liver to establish the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Blood perfusion of tumor in control, laparotomy control, and HAL group was analyzed by Hoechst 33342 labeling assay, the serum VEGF level was assayed by ELISA, the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of tumor directly and hypoxia of tumor indirectly in HAL group decreased significantly compared with that in control group (329+/-29 vs 384+/-19, P【0.01). The serum VEGF level in the HAL group increased significantly as against that of the control group (93 ng.L(-1)+/-44 ng.L(-1) vs 55 ng.L(-1)+/-19 ng.L(-1), P【0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P【0.05). The blood perfusion data of the tumor, represented by the number of Hoechst 33342 labeled cells, showed a good linear inverse correlation with the serum VEGF level (r=-0.606, P【0.05) and the expression of VEGF mRNA in the tumor tissue ( r =-0.338, P【0.01). CONCLUSION: Blockage of hepatic arterial blood supply results in decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major cause of up-regulated expression of VEGF.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Tumoricidal activation of murine resident peritoneal macrophages on pancreatic carcinoma by interleukin-2 and monoclonal antibodies

INTRODUCTIONMacrophages play an important role in tumor lysisand growth inhibition.They can be activated to atumoricidal state by a variety of agents such asIFNr,TNFα or IL2.The killing machanisms ofactivated macrophages have been extensivelyinvestigated.Recently,it has been proved thatantibody dependent cellular cytotoxicity (ADCC) isone of the potent arms to lyse tumor cells

AUGMENTATION OF IMMUNE FUNCTIONS AND AUTOLOGOUS TUMOR-KILLING ACTIVITY BY KAPPA-SELENOCARRAGEENAN IN MICE BEARING SARCOMA 180

Objective: To study the enhancement of the immune functions and autologous tumor killing (ATK) activity by kappa selenocarrageenan (KSC) in mice bearing sarcoma 180. Methods: To measure the effects of KSC and/or Cyclophosphamide (Cy) on natural killer (NK) activity, lymphokine activated killer (LAK) activity, the produc tion of interleukin 2 (IL 2), ATK activity and the growth of sarcoma 180 (S 180 ). Results: KSC promoted NK activity, LAK activity and ATK activity in vivo , increased IL 2 production at 40 mg/kg/d×9d. It also enhanced the antitumor action of Cy (20 mg/kg/d×9d) and offset the inhibition of Cy on immunocopetent cells. The ATK activity in splenocytes of S 180 bearing mice could be induced and increased by recombinant interleukin 2 (rIL 2) in vitro . Conclusion: KSC has an up regulating effect on the immune functions and ATK activity in tumor bearing mice. It can be used as a biological response modifier (BRM) in cancer biotherapy.

Sishengtang decoction in alleviation of toxic and side effects of transarterial embolization

AIM To observe the therapeutic effects of Sishengtang decoction in alleviating the toxic and side effects of transarterial embolization (TAE). METHODS Fifty four patients with liver cancer were divided randomly into Sishengtang decoction group (34 cases) and control group (20 cases). The changes of clinical symptoms and peripheral hemogram and some cellular immune functions were observed before and two weeks after TAE. RESULTS Sishengtang decoction was superior to the control group in improving the digestive tract reaction. The leucocytes of peripheral blood and cellular immune functions (activities of NK cells and LAK cells) of control group decreased obviously after TAE, while that of Sishengtang decoction group decreased slightly, without obvious difference as compared with that of preoperation. CONCLUSIONS Sishengtang decoction might improve the clinical symptoms and increase the leucocytes of peripheral blood and the cellular immune functions of TAE patients.

Clinical Study on General lmmunotherapy of Hepatocellular Carcinoma

In the present study,67 cases of hepatocellular carcinoma(HCC)were treated withimmunotherapy or immunochernotherapy.The results indicated that natural killer cell(NK)andantibody-dependent cytotoxicity cell(ADCC)activities and lymphocyte transformation rate(LTR)were significantly improved in vivo after treatment.The NK activity in tumor tissue,which showed aheavy immunosuppression,could also be enhanced and recovered to normal level after treatment withlymphokine mixture and 5-fluorouracil(5-FU).Immunity of host with postoperativeimmunochemotherapy was rapidly improved and recovery vate was high.The survival time in thesecases of unresectable tumors was obviously prolonged when a general immunotherapy was used.Immunochemotherapy can impr(?)ve immunity and inhibit selectively Ts cells as well as kill directly thetremor cells;therefore,it can play an important role in the treatment of hepatocellular carcinomaand in the prevention of its recurrence after operation.

In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell-replaced murine spleen cell cultures

Specific antibody responses could be induced in serumfree condition.Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium.This induction system may facilitate the study of lymphokine functions on antigen triggered B cells. In T cell-replaced cultures,the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens.It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells.The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.

EFFECT OF KANGLEMYCIN C ON LYMPHOKIN A PRODUCTION AND GENE EXPRESSION OF MOUSE SPLENOCYTE AND MACROPHAGE

Aim. To study the effect of kanglemycin C (KC) on production and gene transcription of lymphokins. Methods.Cell proliferation and lymphokin activities were quantified with MTT colorimetry and ELISA, and gene transcriptions of lymphokins semi quantified with RT PCR. Results.Suppression of KC on proliferation of enriched T and B cell respectively mediated by Con A and LPS was declined by addition of exogenous IL 1, IL 2, and IL 6. KC 80 nmol/L markedly inhibited IL 2 and IL 6 production and mRNA transcription of incubated mouse splenocytes induced by Con A. Additionally, KC had some suppression on IL 1β and IL 6 productions of peritoneal macrophage stimulated by LPS (5μg/mL), whereas cyclosporine (CS) had not. [WT5”BX]Conclusion. [WT5”BZ]Immunosuppression of KC came true partially through the decrease of IL 1β, 2 and 6 productions, especially of IL 2. However, CS′s immunosuppression was mainly through the decrease of IL 2 procduction.

Expression and significance of the vascular permeability factor in nasal polyps

目的探讨血管通透性因子(vascular permeability factor, VPF)在鼻息肉组织中的表达及意义.方法将9例鼻息肉标本及8例下鼻甲粘膜标本行VPF及其受体flk-1的免疫组化染色,光镜观查.结果 VPF在鼻息肉组织的血管内皮细胞和腺体细胞的表达明显高于下鼻甲组织(P<0.01,P<0.05),flk-1在血管内皮细胞的表达明显高于下鼻甲组织(P<0.01).结论 VPF对鼻息肉发生过程中组织极度水肿的产生可能有非常重要的作用.

Vascular endothelial growth factor gene transfer improves host endothelialization of xenogeneic biologic heart valve in vivo

OBJECTIVE: To investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene. METHODS: Bovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously. RESULTS: The concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P

Efficacy and safety of therapeutic angiogenesis from direct myocardial administration of an adenoviral vector expressing vascular endothelial growth factor 165

OBJECTIVE: To investigate whether direct administration of adenoviral vectors (Ad) containing the complementary deoxyribonucleic acid (cDNA) of vascular endothelial growth factor 165 (Ad-VEGF165) induces porcine coronary collateral vessel formation, improves regional myocardial perfusion and function and is safe. METHODS: Three weeks after miniature swine underwent left thoracotomy and placement of an Ameroid constrictor on the left circumflex coronary artery (LCX), Ad-VEGF165 (n = 6) or the control, Ad expressing beta-galactosidase cDNA (Ad-Gal, n = 6), was directly administered into the ischemic myocardium in the circumflex distribution. All animals were sacrificed 4 wk after the second surgery. Myocardial perfusion and function were assessed by electrocardiogram-gated single photon emission computed tomography (GSPECT) imaging. Ex vivo coronary angiography was performed to examine collateral vessels. Toxicity was assessed by blood analyses on the day just before (day 0) and on day 1, 3, 7, 28 after vector delivery and by vascular, myocardial and liver histology after sacrifice. RESULTS: GSPECT imaging 4 wk after administration of Ad-VEGF165 demonstrated significant reduction in ischemic area (P

Extracellular domain of kinase domain region mediated by adeno-associated virus inhibits growth and angiogenesis of bladder cancer in Balb-c mice

OBJECTIVE: To verify whether the extracellular domain of kinase domain region (KDR) has anti-angiogenesis activity in vivo. METHODS: cDNA was cloned into adeno-associated virus (AAV) vector pSNAV and transfected to baby hamster kidney (BHK) cells. Recombinant AAV was obtained from the cell culture supernatant after adding helper virus. Recombinant AAV-infected human bladder cancer EJ cell line (EJ cells) were injected subcutaneously into Balb-c nude mice. Tumor specimens were removed from the mice, paraffin-embedded and sliced, then stained by immunohistochemistry. Microvessel density (MVD) was determined under a microscope. RESULTS: The tumor volume developed by EJ cells transfected with the extracellular domain of KDR was significantly smaller (1.70 +/- 0.18 cm(3)) compared with that in the control (5.62 +/- 0.67 cm(3)) (P

Influence of Flavonoid of Astragalus Membranaceus's Stem and Leaf on the Function of Cell Mediated Immunity in Mice

Objective： To investigate the immune regulation of flavonoid of Astragalus membranaceus's stem and leaf(FAM-sl). Methods： Changes of total T cell count and subsets in mice were determined by monoclonal antibody assay before and after treatment with FAM-sl, and the lymphokine activated killer cell (LAK) activity was tested simultaneously by isotope label method.Results： FAM-sl could promote the proliferation of lymphocytes induced by ConA, raise the total T cell count and regulate the T cell subsets disturbance, and elevate the LAK activity induced by recombinant interleukin-2 (rIL-2).Conclusion： FAM-sl possesses effects of immune stimulation and immune regulation in treating immunosuppressive mice. This study provides experimental evidence for clinical application of FAM-sl.

上期临床病案讨论结果

上期临床病案讨论结果术前诊断左上腹巨大包块：①腹膜后包块（嗜铬细胞瘤？）；②左肝肿瘤？手术情况于１９９４年５月７日在静脉复合麻醉下取双侧肋缘下人字形切口行剖腹探查术。术中见肝脏正常，未扣及结节或肿块，但在左肝下缘与胃小弯之间发现一凸出的巨大肿块。切开...