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Volume 9,Issue 3,2002 Table of Contents
1 in vitro and in vivo Effects of the Targeted Non-Viral Vector GE7 System on Growth of Human Gliomas
CHEN Yong-xin
,
XU Xiu-lan
,
ZHANG Guang-ji
,
WANG Wei
,
ZHAO Rui-jiao
,
JING Hai-ying
,
LU Yi-cheng
,
GU Jian-ren
2002, 9(3):153-157.
DOI: 10.3872/j.issn.1007-385X.2002.3.001
Abstract:
Objective:To investigate in vitro and in vivo effects on growth of human gliomas mediated by the EGF-R targeting non-viral vector GE7 system.Methods:To construct GE7 system, U251MG cell line was transfected in vitro with β-galactosidase gene and p21WAF-1/CIP1 gene mediated by GE7 system. By means of X-gal staining, MTS, FACS, the transferring rate of exogenous gene and the apoptosis rate of tumor cells were examined. The animal model was established by implanting U251 cells subcutaneously in nude mouse. The U251MG glioma-bearing animals were injected with GE7/DNA/HA20 complexes subcutaneously and introvenously. One week later animals were killed, observed the average weight of each group and tumor inhibitory rate in different groups.Results:The highest transferring rate of GE7 system in vitro was 70%,and the expression of p21WAF-1/CIP1 gene could induce the apoptosis of the glioma cell and inhibit its growth. The average apoptosis rate was 25.2%. The subcutaneous and introvenous injection of GE7 system had the same therapeutic effect on human gliomas with the tumor inhibitory rate of 56.5% and 60.2%. Conclusion：This study provides a more effective way to improve the effeciency of glioma gene therapy.
Objective:To investigate in vitro and in vivo effects on growth of human gliomas mediated by the EGF-R targeting non-viral vector GE7 system.Methods:To construct GE7 system, U251MG cell line was transfected in vitro with β-galactosidase gene and p21WAF-1/CIP1 gene mediated by GE7 system. By means of X-gal staining, MTS, FACS, the transferring rate of exogenous gene and the apoptosis rate of tumor cells were examined. The animal model was established by implanting U251 cells subcutaneously in nude mouse. The U251MG glioma-bearing animals were injected with GE7/DNA/HA20 complexes subcutaneously and introvenously. One week later animals were killed, observed the average weight of each group and tumor inhibitory rate in different groups.Results:The highest transferring rate of GE7 system in vitro was 70%,and the expression of p21WAF-1/CIP1 gene could induce the apoptosis of the glioma cell and inhibit its growth. The average apoptosis rate was 25.2%. The subcutaneous and introvenous injection of GE7 system had the same therapeutic effect on human gliomas with the tumor inhibitory rate of 56.5% and 60.2%. Conclusion：This study provides a more effective way to improve the effeciency of glioma gene therapy.
2002, 9(3):158-162.
DOI: 10.3872/j.issn.1007-385X.2002.3.002
Abstract:
Objective: To construct an eukaryotic expressing plasmid of mouse TRAIL (mTRAIL), and investigate its apoptosis-inducing ability to hepatocelluar carcinoma cells in vitro and in vivo, inhibitory effect on the growth of hepatocelluar carcinoma, and its synergism with pCH510, an eukaryotic expressing plasmid of recombinant human FN polypeptide. Methods: The eukaryotic expressing plasmid of mTRAIL was constructed by RT-PCR and DNA recombination techniques. Gene transfection was performed in vitro and in vivo. The apoptosis rate of hepatocelluar carcinoma cells was measured by Flow Cytometry. The apoptosis of hepatocelluar carcinoma cells was also detected by TdT-mediated dUTP nick end labeling (TUNEL) and histochemistry techniques. The inhibitory effect of gene transfection on solid tumor was observed in mice. Results: The cDNA of mTRAIL was amplified by RT-PCR from the RNA of the mouse spleen cells, and cloned into the eukaroytic expressing vector pcDNA3.1. The recombinant plasmid was designated as pX1. The BHK cells transfected with plasmid pX1 could attack H22 hepatocelluar carcinoma cells and induce them into apoptosis. The transfection of plasmid pX1 through injection into mouse muscles could inhibit the growth of hepatocelluar carcinoma by inducing tumor cells into apoptosis. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth. Conclusion: Plamid pX1 could be expressed in cells and in vivo in mouse. The expression of pX1 in vivo and in vitro could induce hepatocelluar carcinoma cells into apoptosis and inhibit the growth of hepatocelluar carcinoma by this mechanism. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth.
Objective: To construct an eukaryotic expressing plasmid of mouse TRAIL (mTRAIL), and investigate its apoptosis-inducing ability to hepatocelluar carcinoma cells in vitro and in vivo, inhibitory effect on the growth of hepatocelluar carcinoma, and its synergism with pCH510, an eukaryotic expressing plasmid of recombinant human FN polypeptide. Methods: The eukaryotic expressing plasmid of mTRAIL was constructed by RT-PCR and DNA recombination techniques. Gene transfection was performed in vitro and in vivo. The apoptosis rate of hepatocelluar carcinoma cells was measured by Flow Cytometry. The apoptosis of hepatocelluar carcinoma cells was also detected by TdT-mediated dUTP nick end labeling (TUNEL) and histochemistry techniques. The inhibitory effect of gene transfection on solid tumor was observed in mice. Results: The cDNA of mTRAIL was amplified by RT-PCR from the RNA of the mouse spleen cells, and cloned into the eukaroytic expressing vector pcDNA3.1. The recombinant plasmid was designated as pX1. The BHK cells transfected with plasmid pX1 could attack H22 hepatocelluar carcinoma cells and induce them into apoptosis. The transfection of plasmid pX1 through injection into mouse muscles could inhibit the growth of hepatocelluar carcinoma by inducing tumor cells into apoptosis. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth. Conclusion: Plamid pX1 could be expressed in cells and in vivo in mouse. The expression of pX1 in vivo and in vitro could induce hepatocelluar carcinoma cells into apoptosis and inhibit the growth of hepatocelluar carcinoma by this mechanism. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth.
2002, 9(3):163-167.
DOI: 10.3872/j.issn.1007-385X.2002.3.003
Abstract:
Objective: To observe effective of antitumor immunity elicited by heat shock protein70(HSP70)-associated peptide complexes isolated from hepatoma (HCaF), a tumor model of no MHC-Ⅰmolecule expression in mice. Methods: Specific active immunization and adoptive cellular immunization assay was adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes purfied from the HCaF.Results: Mice immunized with HSP70-associated peptide complexes were protected from HCaF challenge. The immunoprotective effect in female mice was better than the male groups. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70-associated peptide complexes could be elicited immunity against HCaF challenge, and the mice free of tumor can resist repeated challenge, the maximal challenge-quantities exceeded 1×10 7 HCaF cells. Mice immunized once with spleen cells pulsed by HSP70-associated peptide complexes could also be elicited certain immunity against HCaF challenge. Conclusions: HSP70-associated peptide complexes derived from the HCaF can elicit no-MHC-Ⅰ molecule restrictive immunoprotective effect against HCaF and could be continuously enhanced by repeated challenge with alive HCaF cells. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70-associated peptide complexes could provide protection against HCaF cells challenge.
Objective: To observe effective of antitumor immunity elicited by heat shock protein70(HSP70)-associated peptide complexes isolated from hepatoma (HCaF), a tumor model of no MHC-Ⅰmolecule expression in mice. Methods: Specific active immunization and adoptive cellular immunization assay was adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes purfied from the HCaF.Results: Mice immunized with HSP70-associated peptide complexes were protected from HCaF challenge. The immunoprotective effect in female mice was better than the male groups. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70-associated peptide complexes could be elicited immunity against HCaF challenge, and the mice free of tumor can resist repeated challenge, the maximal challenge-quantities exceeded 1×10 7 HCaF cells. Mice immunized once with spleen cells pulsed by HSP70-associated peptide complexes could also be elicited certain immunity against HCaF challenge. Conclusions: HSP70-associated peptide complexes derived from the HCaF can elicit no-MHC-Ⅰ molecule restrictive immunoprotective effect against HCaF and could be continuously enhanced by repeated challenge with alive HCaF cells. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70-associated peptide complexes could provide protection against HCaF cells challenge.
2002, 9(3):168-171.
DOI: 10.3872/j.issn.1007-385X.2002.3.004
Abstract:
Objective:To investigate the antitumor effects of arsenic trioxide (As2O3) on solid tumor in vivo, the transplanted hepatic carcinoma of mice were treated with various dosages of As2O3. Methods: After 50 mice were injected intraperitoneally with 0，2, 4, 6, 8 μmol/g As2O3 respectively for observation of acute toxicity, 1, 2, 3 μmol/g were selected for this experiment. Eighty mice were transplanted with hepatic carcinoma cells (1.5×106 cells/each side) into both subaxillae. As2O3 in 0, 1, 2 and 3 μmol/g were injected into right subaxilla respectively once every day for 10 days. At the 30th day after transplanted hepatic carcinoma cell, mice were killed and the size of solid tumors was measured. The chronic cytotoxicity of As2O3 was observed in these mice.Results:In right subaxillae tumors, which were injected directly with As2O, were apparent smaller than that in the left (P<0.05). Comparing different dosage, the right solid tumors in 2 and 3 μmol/g groups were smaller than that in control group (P<0.001). In the left the tumor masses of 1 and 2 μmol/g groups were larger than that in control group (P<0.05) and 3 μmol/g group vice versa (P<0.05). The mechanism of action of As2O3 was inducement of cellular apoptosis. The damages of liver, kidney, spleen and decrease of WBC were found by chronic toxicity of As2O3.Conclusion: Our results suggested that As2O3 in a convenient dosage and in direct contact with tumor cells might be a useful, novel therapy for epithelial solid tumor and the permissible dosage and the route of administration were emphasized in the anticancer therapy.
Objective:To investigate the antitumor effects of arsenic trioxide (As2O3) on solid tumor in vivo, the transplanted hepatic carcinoma of mice were treated with various dosages of As2O3. Methods: After 50 mice were injected intraperitoneally with 0，2, 4, 6, 8 μmol/g As2O3 respectively for observation of acute toxicity, 1, 2, 3 μmol/g were selected for this experiment. Eighty mice were transplanted with hepatic carcinoma cells (1.5×106 cells/each side) into both subaxillae. As2O3 in 0, 1, 2 and 3 μmol/g were injected into right subaxilla respectively once every day for 10 days. At the 30th day after transplanted hepatic carcinoma cell, mice were killed and the size of solid tumors was measured. The chronic cytotoxicity of As2O3 was observed in these mice.Results:In right subaxillae tumors, which were injected directly with As2O, were apparent smaller than that in the left (P<0.05). Comparing different dosage, the right solid tumors in 2 and 3 μmol/g groups were smaller than that in control group (P<0.001). In the left the tumor masses of 1 and 2 μmol/g groups were larger than that in control group (P<0.05) and 3 μmol/g group vice versa (P<0.05). The mechanism of action of As2O3 was inducement of cellular apoptosis. The damages of liver, kidney, spleen and decrease of WBC were found by chronic toxicity of As2O3.Conclusion: Our results suggested that As2O3 in a convenient dosage and in direct contact with tumor cells might be a useful, novel therapy for epithelial solid tumor and the permissible dosage and the route of administration were emphasized in the anticancer therapy.
2002, 9(3):172-174.
DOI: 10.3872/j.issn.1007-385X.2002.3.005
Abstract:
Objective： To explore the immune effects and the solid tumor growth of arsenic trioxide on experimental H22 Hepatoma-bearing mice.Methods：T lymphocyte subsets and NKcells activity were assessed by flow cytometry and improved MTT assay. Calculate the tumor inhibitory rates. Results: The percentage of CD3＋ cells and CD4＋ cells, the ratio of CD4+ cells to CD8+ cells, the activity of NK cells in the tumor control group were markedly lower (P<0.01) than those in the healthy control group. However, they were significantly higher in the tumor treatment than in the tumor control group. There was a higher percentage of CD4+ and NKCA in arsenic trioxide treatment group at high dosage than that at low dosage (P<0.05). The tumor inhibitory rates were 39.12% and 45.74% separately. A lot of inflammatory cells could be seen under optic microscopy. Conclusion：Arsenic trioxide can obviously increase the immune function of H22 Hepatoma-bearing mice and inhibit the growth of tumor body.
Objective： To explore the immune effects and the solid tumor growth of arsenic trioxide on experimental H22 Hepatoma-bearing mice.Methods：T lymphocyte subsets and NKcells activity were assessed by flow cytometry and improved MTT assay. Calculate the tumor inhibitory rates. Results: The percentage of CD3＋ cells and CD4＋ cells, the ratio of CD4+ cells to CD8+ cells, the activity of NK cells in the tumor control group were markedly lower (P<0.01) than those in the healthy control group. However, they were significantly higher in the tumor treatment than in the tumor control group. There was a higher percentage of CD4+ and NKCA in arsenic trioxide treatment group at high dosage than that at low dosage (P<0.05). The tumor inhibitory rates were 39.12% and 45.74% separately. A lot of inflammatory cells could be seen under optic microscopy. Conclusion：Arsenic trioxide can obviously increase the immune function of H22 Hepatoma-bearing mice and inhibit the growth of tumor body.
2002, 9(3):175-178.
DOI: 10.3872/j.issn.1007-385X.2002.3.006
Abstract:
Objective: To explore the inhibition of E-selectin and ICAM-1 expression in endothelial cells and the effects on adhesion of endothelial cells to hepatocarcinoma cells by antisense oligodeoxynucleotide（ASODN）.Methods:With treatment of ASODN, the changes of E-selectin and ICAM-1 protein expression in endothelial cells induced by TNF-α were examined by flow cytometry, E-selectin and ICAM-1 mRNA expression by RT-PCR, and the adhesion rate of endothelial cells to hepatoma cells by cell adhesion experiment. Results: Both nude ASODN and liposome-ASODN apparently brought down the expression of E-selectin and ICAM-1 protein, as well as mRNA, and the latter worked better for all that lower dosage. E-selectin ASODN significantly decreased the adhesion rate of endothelial cells to hepatocarcinoma cells.Conclusion: These data demonstratel that ASODNs were capable of selectively inhibiting the expression of E-selectin and ICAM-1 in endothelial cells and thereby, to a certain extent, reducing the adhesion between endothelial cells and hepatocarcinoma cells.
Objective: To explore the inhibition of E-selectin and ICAM-1 expression in endothelial cells and the effects on adhesion of endothelial cells to hepatocarcinoma cells by antisense oligodeoxynucleotide（ASODN）.Methods:With treatment of ASODN, the changes of E-selectin and ICAM-1 protein expression in endothelial cells induced by TNF-α were examined by flow cytometry, E-selectin and ICAM-1 mRNA expression by RT-PCR, and the adhesion rate of endothelial cells to hepatoma cells by cell adhesion experiment. Results: Both nude ASODN and liposome-ASODN apparently brought down the expression of E-selectin and ICAM-1 protein, as well as mRNA, and the latter worked better for all that lower dosage. E-selectin ASODN significantly decreased the adhesion rate of endothelial cells to hepatocarcinoma cells.Conclusion: These data demonstratel that ASODNs were capable of selectively inhibiting the expression of E-selectin and ICAM-1 in endothelial cells and thereby, to a certain extent, reducing the adhesion between endothelial cells and hepatocarcinoma cells.
2002, 9(3):179-182.
DOI: 10.3872/j.issn.1007-385X.2002.3.007
Abstract:
Objective: To investigate the inhibitory effects of the cytokine-induced killer (CIK) cells from human peripheral blood mononuclear cells (PBMCs) origin on the growth of hepatocellular carcinoma (HCC) cells in animal model. Methods: PBMCs from healthy donors were enriched by using the specific program of the Cobe Spectra blood separator and induced in vitro into CIK cells in serum-free culture medium containing interferon-gamma (IFN-γ), interleukin-2 (IL-2)，and anti-CD3. The phenotypes and characterization of CIK cells were identified by flow cytometric analysis. BALB/c nude mice subscapularly transplanted with 1.5×105 of BEL-7402 HCC cell at the exponential growth produced 100% tumor incidence and were treated with human CIK cells for consecutive six days on the second day after HCC transplantation. Results:The CIK cells identified by flow cytometric analyses were shown to be a heterogeneous population with different cellular phenotypes. The total CIK cells and CD3+/CD56+ positive cells significantly increased by 7 fold and 6-fold, respectively, in cell proliferation number at day 13 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells. In tumor-bearing nude mice, human CIK cells showed a significant inhibitory effect on the growth of transplanted HCC, resulting in a statistical significant difference among the treated and control groups. There was dose-response dependent relationship between the inhibitory effect and number of treated CIK cells. Conclusion: Human CIK cells are of highly efficient cytotoxic effector cells against hepatocellular carcinoma cells in murine model and are likely to be used as an immune therapeutic strategy for HCC patients
Objective: To investigate the inhibitory effects of the cytokine-induced killer (CIK) cells from human peripheral blood mononuclear cells (PBMCs) origin on the growth of hepatocellular carcinoma (HCC) cells in animal model. Methods: PBMCs from healthy donors were enriched by using the specific program of the Cobe Spectra blood separator and induced in vitro into CIK cells in serum-free culture medium containing interferon-gamma (IFN-γ), interleukin-2 (IL-2)，and anti-CD3. The phenotypes and characterization of CIK cells were identified by flow cytometric analysis. BALB/c nude mice subscapularly transplanted with 1.5×105 of BEL-7402 HCC cell at the exponential growth produced 100% tumor incidence and were treated with human CIK cells for consecutive six days on the second day after HCC transplantation. Results:The CIK cells identified by flow cytometric analyses were shown to be a heterogeneous population with different cellular phenotypes. The total CIK cells and CD3+/CD56+ positive cells significantly increased by 7 fold and 6-fold, respectively, in cell proliferation number at day 13 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells. In tumor-bearing nude mice, human CIK cells showed a significant inhibitory effect on the growth of transplanted HCC, resulting in a statistical significant difference among the treated and control groups. There was dose-response dependent relationship between the inhibitory effect and number of treated CIK cells. Conclusion: Human CIK cells are of highly efficient cytotoxic effector cells against hepatocellular carcinoma cells in murine model and are likely to be used as an immune therapeutic strategy for HCC patients
2002, 9(3):183-185.
DOI: 10.3872/j.issn.1007-385X.2002.3.008
Abstract:
Objective: To investigate the characteristics of the cultured cervical cancer cell line transfected with anti-HPV16E6-ribozyme, and to investigate the possibility and practicality of ribozyme in treatment of cervical cancer. Methods:The anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected by lipofectin transfection into CaSKi cell, which named as CaSKi-R, CaSKi-P respectively. The morphology and the soft agar forming ability were studied. The expression of E6, PCNA and C-erbB-2 genes was studied through Flow Cytometry. The tumorgenicity of each cell was detected by injecting cells into the nude mice skin. Three groups of nude mice were injected by CaSKi, CaSKi-R and CaSKi-P cell separately. Another group of mice was injected by CaSKi cell on right side and CaSKi-R cell on left side. Results: There is no distinct difference of the morphology and growth rate between CaSKi and CaSKi-P, but the growth rate of CaSKi-R decreased. The soft agar-forming rate of CaSKi-P was similar with that of CaSKi cells, while that of CaSKi-R was found decreased. The result of flow cytometric analysis showed that anti-HPV16E6-ribozyme could reduce the expression of E6, PCNA and C-erbB-2 genes on CaSKi-R cells, while this phenomenon was not found on the CaSKi-P cells. The tumorgenicity of CaSKi-R in nude mice was decreased compared with CaSKi and CaSKi-P cells. Conclusion: Anti-HPVE6-rivozyme could partly reverse the malignant phenotypes of CaSKi cells. The reason may be the decrease of E6 gene expression, and the succeeding decrease of the PCNA and C-erbB-2 genes′ expression.
Objective: To investigate the characteristics of the cultured cervical cancer cell line transfected with anti-HPV16E6-ribozyme, and to investigate the possibility and practicality of ribozyme in treatment of cervical cancer. Methods:The anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected by lipofectin transfection into CaSKi cell, which named as CaSKi-R, CaSKi-P respectively. The morphology and the soft agar forming ability were studied. The expression of E6, PCNA and C-erbB-2 genes was studied through Flow Cytometry. The tumorgenicity of each cell was detected by injecting cells into the nude mice skin. Three groups of nude mice were injected by CaSKi, CaSKi-R and CaSKi-P cell separately. Another group of mice was injected by CaSKi cell on right side and CaSKi-R cell on left side. Results: There is no distinct difference of the morphology and growth rate between CaSKi and CaSKi-P, but the growth rate of CaSKi-R decreased. The soft agar-forming rate of CaSKi-P was similar with that of CaSKi cells, while that of CaSKi-R was found decreased. The result of flow cytometric analysis showed that anti-HPV16E6-ribozyme could reduce the expression of E6, PCNA and C-erbB-2 genes on CaSKi-R cells, while this phenomenon was not found on the CaSKi-P cells. The tumorgenicity of CaSKi-R in nude mice was decreased compared with CaSKi and CaSKi-P cells. Conclusion: Anti-HPVE6-rivozyme could partly reverse the malignant phenotypes of CaSKi cells. The reason may be the decrease of E6 gene expression, and the succeeding decrease of the PCNA and C-erbB-2 genes′ expression.
2002, 9(3):186-189.
DOI: 10.3872/j.issn.1007-385X.2002.3.009
Abstract:
目的: 研究降钙素对人乳腺癌细胞系T47D的作用。方法: 将鲑鱼降钙素(sCT)作用于体外培养的T47D细胞,MTT法观察细胞生长抑制率, PCR-ELISA法分析细胞端粒酶活性,透射电镜观察细胞凋亡。将T47D细胞接种至裸鼠皮下并肌注sCT,30 d后测量瘤体直径,扫描电镜比较第三腰椎的结构。结果: sCT能抑制T47D细胞的生长,降低端粒酶活性,诱导细胞凋亡。体内试验未观察到瘤体缩小,扫描电镜发现sCT使第三腰椎骨质结构增粗致密。 结论: 降钙素诱导T47D细胞凋亡并降低其端粒酶活性是其抑制乳腺癌细胞体外生长的新机制。体内试验未得到理想的抑瘤效果,可能与机体复杂的内分泌反应有关。对于无骨质疏松的裸鼠,降钙素亦能发挥促骨质沉积作用。
目的: 研究降钙素对人乳腺癌细胞系T47D的作用。方法: 将鲑鱼降钙素(sCT)作用于体外培养的T47D细胞,MTT法观察细胞生长抑制率, PCR-ELISA法分析细胞端粒酶活性,透射电镜观察细胞凋亡。将T47D细胞接种至裸鼠皮下并肌注sCT,30 d后测量瘤体直径,扫描电镜比较第三腰椎的结构。结果: sCT能抑制T47D细胞的生长,降低端粒酶活性,诱导细胞凋亡。体内试验未观察到瘤体缩小,扫描电镜发现sCT使第三腰椎骨质结构增粗致密。 结论: 降钙素诱导T47D细胞凋亡并降低其端粒酶活性是其抑制乳腺癌细胞体外生长的新机制。体内试验未得到理想的抑瘤效果,可能与机体复杂的内分泌反应有关。对于无骨质疏松的裸鼠,降钙素亦能发挥促骨质沉积作用。
10 The Effect of Antibody of Vascular Endotheial Growth Factor on Ovarian Cancer Line SKOV3 in vitro
2002, 9(3):190-193.
DOI: 10.3872/j.issn.1007-385X.2002.3.010
Abstract:
Objective:To explore the effect of antibody of vascular endothelial growth factor (VEGF)on ovarian cancer line SKOV3. Methods:The different concentrations of anti-VEGF-antibody was used to act on SKOV3 in vitro. The RT-PCR and immunohistochemically methods were used to determine mRNA and protein expression of VEGF in SKOV3. The VEGF content of SKOV3 in culture medium was test by ELISA. The MTT and cell counting were also used to determine the growth curve and inhibited rate of SKOV3 under different concentrations of anti-VEGF-antibody. Results: (1) After the different concentrate of anti-VEGF-antibody was used to act on SKOV3 in vitro, there is not significant difference in inhibition rate and proliferation rate compared with the control (P>0.05). (2) VEGF mRNA, protein and VEGF-receptor expression of SKOV3 treated with anti-VEGF-antibody is down-regulated. (3) The VEGF content of SKOV3 in culture medium treated with anti-VEGF-antibody is lower than that in control (P<0.05). Conclusions: VEGF antibody had no influence on growth of SKOV3 in vitro, but it could downregulate the expression of VEGF and it′s receptor and inhibit the secretion of VEGF in SKOV3.
Objective:To explore the effect of antibody of vascular endothelial growth factor (VEGF)on ovarian cancer line SKOV3. Methods:The different concentrations of anti-VEGF-antibody was used to act on SKOV3 in vitro. The RT-PCR and immunohistochemically methods were used to determine mRNA and protein expression of VEGF in SKOV3. The VEGF content of SKOV3 in culture medium was test by ELISA. The MTT and cell counting were also used to determine the growth curve and inhibited rate of SKOV3 under different concentrations of anti-VEGF-antibody. Results: (1) After the different concentrate of anti-VEGF-antibody was used to act on SKOV3 in vitro, there is not significant difference in inhibition rate and proliferation rate compared with the control (P>0.05). (2) VEGF mRNA, protein and VEGF-receptor expression of SKOV3 treated with anti-VEGF-antibody is down-regulated. (3) The VEGF content of SKOV3 in culture medium treated with anti-VEGF-antibody is lower than that in control (P<0.05). Conclusions: VEGF antibody had no influence on growth of SKOV3 in vitro, but it could downregulate the expression of VEGF and it′s receptor and inhibit the secretion of VEGF in SKOV3.
2002, 9(3):194-197.
DOI: 10.3872/j.issn.1007-385X.2002.3.011
Abstract:
Objective: To clone human angiogenesis inhibitor restin (hRS), express fusion protein in E.Coli and determine its biological activity. Methods: Restin gene was amplified by RT-PCR from Chinese human placenta tissue, then inserted into plasmid vector pGEM-T and sequenced. Prokaryotic expression vector pGEX-hRS was constructed and fusion protein GST-hRS was expressed. After the fusion protein purified by affinity chromatography and digested by thrombin, the anti-angiogenic activity of restin was tested by chicken chorio-allantoic assay. Results: RT-PCR product is 564 bp, the result of DNA sequencing identified the PCR product with the cDNA encoded human restin (Genbank COL15A1), but the synonymous mutation in bases encoding Ser21 (TCT→TCG) and mutation in Ser→Thr82(ACA→TCA) were also discovered. The expressed protein size was 20 kD after isolated fusion protein digested by thrombin, it appeared the expressed restin had the power to inhibit angiogenesis. Conclusion: The successful cloning and expression of human restin lay the foundation for the therapy of solid tumors.
Objective: To clone human angiogenesis inhibitor restin (hRS), express fusion protein in E.Coli and determine its biological activity. Methods: Restin gene was amplified by RT-PCR from Chinese human placenta tissue, then inserted into plasmid vector pGEM-T and sequenced. Prokaryotic expression vector pGEX-hRS was constructed and fusion protein GST-hRS was expressed. After the fusion protein purified by affinity chromatography and digested by thrombin, the anti-angiogenic activity of restin was tested by chicken chorio-allantoic assay. Results: RT-PCR product is 564 bp, the result of DNA sequencing identified the PCR product with the cDNA encoded human restin (Genbank COL15A1), but the synonymous mutation in bases encoding Ser21 (TCT→TCG) and mutation in Ser→Thr82(ACA→TCA) were also discovered. The expressed protein size was 20 kD after isolated fusion protein digested by thrombin, it appeared the expressed restin had the power to inhibit angiogenesis. Conclusion: The successful cloning and expression of human restin lay the foundation for the therapy of solid tumors.
GU Tao
,
XI Hong
,
ZHUANG Yu-mei
,
ZHU Yi-bei
,
QIU Yu-hua
,
QI Chun-jian
,
YU Ge-hua
,
LI Min
,
MA Hong-bing
,
ZHANG Xue-guang
2002, 9(3):198-202.
DOI: 10.3872/j.issn.1007-385X.2002.3.012
Abstract:
Objective: To establish the secreting idiotype immunogloblin (Id) BALB/c mouse B cell lymphoma animal model for further study of Id-dendritic cells (DC) vaccines. Methods: BALB/c mouse B cell lymphoma cell lines were obtained by fusing SP2/0 cells with BALB/c mouse spleen cells after immunization with pristane and incomplete Freund s adjuvant (IFA) respectively. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to screen the secreting Id clones and analysis the phenotypes. Both mice ascites induction and serum-free culture medium X-VIVO 20 were used to produce the Id of SB4 and SB5. By using euglobulin precipitation and gel filtration on Suphecray-200 to purify the Id. To assess the ability of tumorigenicity, （4～5）×10 5 SB4/5 cells were injected subcutaneously in the right axillary or into abdominal cavity of 6~8 week BALB/c mice. DC were induced from bone marrow progenitor cells of B cell lymphoma bearing mice with recombinant murine GM-CSF and IL-4. Results: Two secreting none auto-reactive Id malignant B cell lymphoma cell lines, named as SB4 and SB5, were screened. Chromosomes study, DNA index, cell cycle proliferative indexes, cell generation time and mitotic index analyses all demonstrated SB4 and SB5 possessing the characteristics of malignant tumor cell lines. Both Id proteins belonged to IgM/κ. SB4 and SB5 cells grew progressively in syngeneic mice. Tumors could be touched in all mice and the average life-span was 30± 4 days and typical DC could be induced. Conclusion: BALB/c mouse B cell lymphoma SB4 and SB5 cell lines created by cell fusion techniques could secrete non-auto-reactive Id and had the characteristics of B cell lymphoma cells.
Objective: To establish the secreting idiotype immunogloblin (Id) BALB/c mouse B cell lymphoma animal model for further study of Id-dendritic cells (DC) vaccines. Methods: BALB/c mouse B cell lymphoma cell lines were obtained by fusing SP2/0 cells with BALB/c mouse spleen cells after immunization with pristane and incomplete Freund s adjuvant (IFA) respectively. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to screen the secreting Id clones and analysis the phenotypes. Both mice ascites induction and serum-free culture medium X-VIVO 20 were used to produce the Id of SB4 and SB5. By using euglobulin precipitation and gel filtration on Suphecray-200 to purify the Id. To assess the ability of tumorigenicity, （4～5）×10 5 SB4/5 cells were injected subcutaneously in the right axillary or into abdominal cavity of 6~8 week BALB/c mice. DC were induced from bone marrow progenitor cells of B cell lymphoma bearing mice with recombinant murine GM-CSF and IL-4. Results: Two secreting none auto-reactive Id malignant B cell lymphoma cell lines, named as SB4 and SB5, were screened. Chromosomes study, DNA index, cell cycle proliferative indexes, cell generation time and mitotic index analyses all demonstrated SB4 and SB5 possessing the characteristics of malignant tumor cell lines. Both Id proteins belonged to IgM/κ. SB4 and SB5 cells grew progressively in syngeneic mice. Tumors could be touched in all mice and the average life-span was 30± 4 days and typical DC could be induced. Conclusion: BALB/c mouse B cell lymphoma SB4 and SB5 cell lines created by cell fusion techniques could secrete non-auto-reactive Id and had the characteristics of B cell lymphoma cells.
联系方式
- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
- 国内定价： ￥20元/册
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.
Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.