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Volume 13,Issue 4,2006 Table of Contents
2006, 13(4):239-242.
DOI: 10.3872/j.issn.1007-385X.2006.4.001
Abstract:
2 Anti-tumor effects of murine T-lymphocytes harboring p185HER2 specific chimeric T-cell receptor gene
2006, 13(4):243-248.
DOI: 10.3872/j.issn.1007-385X.2006.4.002
Abstract:
Objective： To investigate the anti-tumor effects of murine T-lymphocytes harboring p185HER2 specific chimeric T-cell receptor gene.Methods: The p185HER2 specific chimeric T-cell receptor N29γ or N29ζ were introduced into retroviral vector pRET6, and the viral producer cell line was established using a Ping-Pong method. Murine spleen T-lymphocytes were transfected using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28, followed by infection in the presence of Retronectin. Transduced murine T-lymphocytes were co-cultured with tumor cell lines overexpressing (SK-OV-3) or underexpressing (MCF-7) p185HER2 for assaying antigen specific cytokine release and CTL. Results: Endonuclease digestion showed the constructed vector was corrected. The titer of retroviral supernatant collected was 1.2×106 and the retroviral transduction efficiency reached over 50% with our optimized method. Both N29γ and N29ζ chimeric T-cell receptor transduced T-lymphocytes demonstrated HER2-specific antigen response as determined by release of interferon γ and cellular cytotoxicity assays. Conclusion: Our results suggest that murine T-lymphocytes harboring chimeric T-cell receptor gene had obvious antitumor effect in vitro through releasing cytokines and CTL effect.
Objective： To investigate the anti-tumor effects of murine T-lymphocytes harboring p185HER2 specific chimeric T-cell receptor gene.Methods: The p185HER2 specific chimeric T-cell receptor N29γ or N29ζ were introduced into retroviral vector pRET6, and the viral producer cell line was established using a Ping-Pong method. Murine spleen T-lymphocytes were transfected using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28, followed by infection in the presence of Retronectin. Transduced murine T-lymphocytes were co-cultured with tumor cell lines overexpressing (SK-OV-3) or underexpressing (MCF-7) p185HER2 for assaying antigen specific cytokine release and CTL. Results: Endonuclease digestion showed the constructed vector was corrected. The titer of retroviral supernatant collected was 1.2×106 and the retroviral transduction efficiency reached over 50% with our optimized method. Both N29γ and N29ζ chimeric T-cell receptor transduced T-lymphocytes demonstrated HER2-specific antigen response as determined by release of interferon γ and cellular cytotoxicity assays. Conclusion: Our results suggest that murine T-lymphocytes harboring chimeric T-cell receptor gene had obvious antitumor effect in vitro through releasing cytokines and CTL effect.
2006, 13(4):249-252.
DOI: 10.3872/j.issn.1007-385X.2006.4.003
Abstract:
Objective: To study the effect of radiotherapy (RT) on dendritic cell (DC)-based immunotherapy of renal cancer in mice. Methods: Mice s.c. renal carcinoma models were established by transplanting Renca renal carcinoma cells and were divided into control group, single radiotherapy group, single DC injection group, and DC injection plus radiotherapy group. Mice in the last group received radiotherapy (7 Gy/time) on d 12-16 after inoculated with renal carcinoma cells (2.5×10 6); and on d 11, 15, 19 and 22, bone marrow-derived un-pulsed DCs (without tumor antigen,10 6 every time) were injected into the tumors of model mice. Mice were sacrificed on day 28 and the growth rate, weight and size of tumors were measured. IL-2, IFN-γ, IL-4 and IL-10 in the splenocytes were determined by ELISA assay.Results: Compared with single DCs and single RT group, DCs plus RT group had an obviously decreased growth rate and tumor size, but the splenocytes had an increased secretion of IL-2, IFN-γ and IL-4 compared with other groups (P<001). Conclusion: Intra-tumor DCs injection combined with radiotherapy can effectively inhibit the growth of renal carcinoma growth in BALB/c mice; its effect is better than any of the single strategy.
Objective: To study the effect of radiotherapy (RT) on dendritic cell (DC)-based immunotherapy of renal cancer in mice. Methods: Mice s.c. renal carcinoma models were established by transplanting Renca renal carcinoma cells and were divided into control group, single radiotherapy group, single DC injection group, and DC injection plus radiotherapy group. Mice in the last group received radiotherapy (7 Gy/time) on d 12-16 after inoculated with renal carcinoma cells (2.5×10 6); and on d 11, 15, 19 and 22, bone marrow-derived un-pulsed DCs (without tumor antigen,10 6 every time) were injected into the tumors of model mice. Mice were sacrificed on day 28 and the growth rate, weight and size of tumors were measured. IL-2, IFN-γ, IL-4 and IL-10 in the splenocytes were determined by ELISA assay.Results: Compared with single DCs and single RT group, DCs plus RT group had an obviously decreased growth rate and tumor size, but the splenocytes had an increased secretion of IL-2, IFN-γ and IL-4 compared with other groups (P<001). Conclusion: Intra-tumor DCs injection combined with radiotherapy can effectively inhibit the growth of renal carcinoma growth in BALB/c mice; its effect is better than any of the single strategy.
CHU Yi-wei
,
LIU Rong-jun
,
ZHANG Lei
,
JIN Mei-ling
,
ZHENG Xiu-juan
,
ZHENG Hui-ru
,
XIONG Si-dong
2006, 13(4):253-256.
DOI: 10.3872/j.issn.1007-385X.2006.4.004
Abstract:
Objective: To investigate the changes of lymphocytes number, proportions of lymphocyte subsets, phenotypes and the cytotoxic function of lymphocytes in lung cancer patients before and after chemotherapy and to assess its clinical relevance. Methods: White blood cells (WBC), lymphocytes and neutrophils were counted before and after chemotherapy in 23 patients with lung cancer. Flow cytometery technique was used to determine and analyze the proportions and phenotypes of CD3+, CD4+ and CD8+ lymphocyte subsets. Memory-like phenotype and cytotoxic function of lymphocytes against K562 cells were detected after peripheral lymphocytes were simulated with PHA for 48 h. Results: Lymphopenia setting (decreased and ensuing recovered cell numbers of WBC, neutrophils and lymphocytes) was observed after chemotherapy. The proportion of CD3+ T cells and the ratio of CD8+/CD4+ were slightly increased and the memory-like phenotype, CD44high and CD62Llow, were exhibited after chemotherapy. The cytotoxicity of lymphocytes against K562 target cells was not decreased, but increased in some patients. Conclusion: Chemotherapy does not hamper, but improve the immune environment in lung cancer patients, which provides a reference for clinical immunotherapy after chemotherapy.
Objective: To investigate the changes of lymphocytes number, proportions of lymphocyte subsets, phenotypes and the cytotoxic function of lymphocytes in lung cancer patients before and after chemotherapy and to assess its clinical relevance. Methods: White blood cells (WBC), lymphocytes and neutrophils were counted before and after chemotherapy in 23 patients with lung cancer. Flow cytometery technique was used to determine and analyze the proportions and phenotypes of CD3+, CD4+ and CD8+ lymphocyte subsets. Memory-like phenotype and cytotoxic function of lymphocytes against K562 cells were detected after peripheral lymphocytes were simulated with PHA for 48 h. Results: Lymphopenia setting (decreased and ensuing recovered cell numbers of WBC, neutrophils and lymphocytes) was observed after chemotherapy. The proportion of CD3+ T cells and the ratio of CD8+/CD4+ were slightly increased and the memory-like phenotype, CD44high and CD62Llow, were exhibited after chemotherapy. The cytotoxicity of lymphocytes against K562 target cells was not decreased, but increased in some patients. Conclusion: Chemotherapy does not hamper, but improve the immune environment in lung cancer patients, which provides a reference for clinical immunotherapy after chemotherapy.
GUO Wen-jing
,
QU Xun
,
ZHANG Bin-bin
,
SHAO Qian-qian
,
GAO Wen-juan
,
ZHENG Guang-juan
,
KONG Bei-hua
2006, 13(4):257-260.
DOI: 10.3872/j.issn.1007-385X.2006.4.005
Abstract:
Objective： To investigate the effect of hypoxia on the mRNA expression of tissue inhibitor of metalloproteinases (TIMPs) in breast carcinoma cell line (MDA-231) and the effect of basil polysaccharide (BP) on the expression of TIMPs. Methods： MDA-231 cells were cultured under normoxia（21% O2, 5% CO2）, hypoxia（1% O2, 5% CO2 and 94% N2）or were treated with BP for 6 h separately. The expression of TIMP1, 2, 3 mRNA was detected by RT-PCR. Results： Expression of TIMP1, 2 mRNA, but not TIMP3 mRNA, was detected in MDA-231 cells. After cultured in hypoxia condition for 6 h, the expression of TIMP1 and TIMP2 mRNA increased significantly (P<0.05). The expression of TIMPs mRNA changed significantly in both hypoxia and normoxia group after BP treatment: the expression of TIMP1, 2 mRNA in normoxia group decreased significantly (P<0.05) and that in hypoxia increased significantly (P<0.05). Conclusion： BP has opposite influence on expression of TIMP1, 2 in human breast cancer cell line MDA-231 under hypoxia and normoxia conditions, indicating that in vitro study on anti-tumor drugs and bio-therapy should take the oxygen environment into consideration.
Objective： To investigate the effect of hypoxia on the mRNA expression of tissue inhibitor of metalloproteinases (TIMPs) in breast carcinoma cell line (MDA-231) and the effect of basil polysaccharide (BP) on the expression of TIMPs. Methods： MDA-231 cells were cultured under normoxia（21% O2, 5% CO2）, hypoxia（1% O2, 5% CO2 and 94% N2）or were treated with BP for 6 h separately. The expression of TIMP1, 2, 3 mRNA was detected by RT-PCR. Results： Expression of TIMP1, 2 mRNA, but not TIMP3 mRNA, was detected in MDA-231 cells. After cultured in hypoxia condition for 6 h, the expression of TIMP1 and TIMP2 mRNA increased significantly (P<0.05). The expression of TIMPs mRNA changed significantly in both hypoxia and normoxia group after BP treatment: the expression of TIMP1, 2 mRNA in normoxia group decreased significantly (P<0.05) and that in hypoxia increased significantly (P<0.05). Conclusion： BP has opposite influence on expression of TIMP1, 2 in human breast cancer cell line MDA-231 under hypoxia and normoxia conditions, indicating that in vitro study on anti-tumor drugs and bio-therapy should take the oxygen environment into consideration.
2006, 13(4):261-265.
DOI: 10.3872/j.issn.1007-385X.2006.4.006
Abstract:
Objective: To investigate the effect of a long-term CpG-ODN stimulation on the maturation of murine bone-marrow derived dendritic cells (BMDCs). Methods: Murine bone-marrow cells were cultured in GM-CSF alone or with CpG-ODN for 7 d or for last 36 h (days 6, 7). Cell phenotypes and antigens uptake by BMDCs were analyzed by flow cytometry. Cytokines released by BMDCs were detected by ELISA. The antigen presenting capability by BMDCs was evaluated by mixed lymphocyte responses.Results:Compared to those of the short-term CpG-ODN stimulation group, the expression of MHCⅡ, CD86, CD40, and secretion of IL-12(p70) by BMDCs in long-term stimulation group were not increased. The phagocytosis of FITC-OVA by BMDCs in long-term CpG-ODN stimulation group was strengthened, but the activation of allogenic and homogenic lymphocyte cells proliferation was impaired. Conclusion:Long-term CpG-ODN stimulation can suppress the maturation of DCs, which may explain the low adaptive immunity in sepsis patients.
Objective: To investigate the effect of a long-term CpG-ODN stimulation on the maturation of murine bone-marrow derived dendritic cells (BMDCs). Methods: Murine bone-marrow cells were cultured in GM-CSF alone or with CpG-ODN for 7 d or for last 36 h (days 6, 7). Cell phenotypes and antigens uptake by BMDCs were analyzed by flow cytometry. Cytokines released by BMDCs were detected by ELISA. The antigen presenting capability by BMDCs was evaluated by mixed lymphocyte responses.Results:Compared to those of the short-term CpG-ODN stimulation group, the expression of MHCⅡ, CD86, CD40, and secretion of IL-12(p70) by BMDCs in long-term stimulation group were not increased. The phagocytosis of FITC-OVA by BMDCs in long-term CpG-ODN stimulation group was strengthened, but the activation of allogenic and homogenic lymphocyte cells proliferation was impaired. Conclusion:Long-term CpG-ODN stimulation can suppress the maturation of DCs, which may explain the low adaptive immunity in sepsis patients.
2006, 13(4):266-271.
DOI: 10.3872/j.issn.1007-385X.2006.4.007
Abstract:
Objective：To construct a recombinant adenovirus (abbreviated as ET-M9-PEX) containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods: Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane (CAM) assay in vivo. Results:ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and gradeⅠvessel number were reduced by 57.57%（P<0.01）and 33.52%（P<0.01）, respectively, compared with E-T. However, neither the tumor weight nor the vessel number was significantly different between E-T and PBS groups.Conclusion: ET-M9-PEX constructed in the present study shows remarkable inhibitory effects on EC proliferation, tumor growth and angiogenesis on CAM, suggesting that ET-M9-PEX may be a promising candidate for tumor gene therapy.
Objective：To construct a recombinant adenovirus (abbreviated as ET-M9-PEX) containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods: Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane (CAM) assay in vivo. Results:ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and gradeⅠvessel number were reduced by 57.57%（P<0.01）and 33.52%（P<0.01）, respectively, compared with E-T. However, neither the tumor weight nor the vessel number was significantly different between E-T and PBS groups.Conclusion: ET-M9-PEX constructed in the present study shows remarkable inhibitory effects on EC proliferation, tumor growth and angiogenesis on CAM, suggesting that ET-M9-PEX may be a promising candidate for tumor gene therapy.
2006, 13(4):272-276.
DOI: 10.3872/j.issn.1007-385X.2006.4.008
Abstract:
Objective： To investigate the influence of BH3-HIV-TAT peptide on proliferation of prostate cancer cell line LNCap.Methods: LNCap cells were treated with BH3-HIV-TAT (25, 50, and 100 μmol/L) for 12 or 24 h. Then, laser scanning confocal fluorescence microscope was used to observe the cellular localization of BH3-HIV-TAT; Hoechst33258 staining was used to observe the morphological changes of LNCap cells during apoptosis; flow cytometry (FCM) was applied to study cell apoptosis and cell cycle of LNCap cells; and MTS/PMS assay was used to analyze whether the effect of BH3-HIV-TAT peptide was concentration dependent.Results: 〖WTBZ〗It was found that BH3-HIV-TAT peptide was mainly located in cell nucleus. The apoptosis of LNCap cells had 2 stages after BH3-HIV-TAT peptide treatment. Flow cytometry confirmed the peptide arrested cell cycle and induced apoptosis of LNCap cells. MTS/PMS assay showed BH3-HIV-TAT peptide had a concentration-dependent anti-LNCap cell effect. Conclusion: BH3-HIV-TAT peptide can effectively induce apoptosis of LNCap cells, influencing the proliferation of the cells, which provide an evidence for targeted therapy of cancer.
Objective： To investigate the influence of BH3-HIV-TAT peptide on proliferation of prostate cancer cell line LNCap.Methods: LNCap cells were treated with BH3-HIV-TAT (25, 50, and 100 μmol/L) for 12 or 24 h. Then, laser scanning confocal fluorescence microscope was used to observe the cellular localization of BH3-HIV-TAT; Hoechst33258 staining was used to observe the morphological changes of LNCap cells during apoptosis; flow cytometry (FCM) was applied to study cell apoptosis and cell cycle of LNCap cells; and MTS/PMS assay was used to analyze whether the effect of BH3-HIV-TAT peptide was concentration dependent.Results: 〖WTBZ〗It was found that BH3-HIV-TAT peptide was mainly located in cell nucleus. The apoptosis of LNCap cells had 2 stages after BH3-HIV-TAT peptide treatment. Flow cytometry confirmed the peptide arrested cell cycle and induced apoptosis of LNCap cells. MTS/PMS assay showed BH3-HIV-TAT peptide had a concentration-dependent anti-LNCap cell effect. Conclusion: BH3-HIV-TAT peptide can effectively induce apoptosis of LNCap cells, influencing the proliferation of the cells, which provide an evidence for targeted therapy of cancer.
2006, 13(4):277-280.
DOI: 10.3872/j.issn.1007-385X.2006.4.009
Abstract:
Objective: To investigate the regulatory effect of interferon-γ on the expression of Fas/FasL and the apoptosis of leukemic cells. Methods: The expression of Fas and its function in human acute leukemic cell line HL-60 and in bone marrow mononuclear cells (BMMNC) of patients with acute myeloid leukemia (AML) were studied by immunohistochemical method, TUNEL method, and cell culture technique. The regulatory effect of interferon-γ on them was also investigated. Results: Compared with BMMNC of normal human, leukemic cells had an obviously decreased expression of Fas and induced apoptosis of Jurkat cells. It was found that interferon-γ up-regulated the expression of Fas in a time- and dose-dependent manner（P<0.01）. Meanwhile, interferon-γ inhibited Jurkat cells apoptosis induced by leukemic cells, increased the Fas-mediated apoptosis of leukemic cells, and increased the sensitivity of leukemic cells to chemotherapy agents.Conclusion: Interferon-γ can prevent the immune escape of leukemic cells via regulating the Fas/FasL system and increase the sensitivity of leukemic cells to the chemotherapy drugs targeting Fas/FasL system.
Objective: To investigate the regulatory effect of interferon-γ on the expression of Fas/FasL and the apoptosis of leukemic cells. Methods: The expression of Fas and its function in human acute leukemic cell line HL-60 and in bone marrow mononuclear cells (BMMNC) of patients with acute myeloid leukemia (AML) were studied by immunohistochemical method, TUNEL method, and cell culture technique. The regulatory effect of interferon-γ on them was also investigated. Results: Compared with BMMNC of normal human, leukemic cells had an obviously decreased expression of Fas and induced apoptosis of Jurkat cells. It was found that interferon-γ up-regulated the expression of Fas in a time- and dose-dependent manner（P<0.01）. Meanwhile, interferon-γ inhibited Jurkat cells apoptosis induced by leukemic cells, increased the Fas-mediated apoptosis of leukemic cells, and increased the sensitivity of leukemic cells to chemotherapy agents.Conclusion: Interferon-γ can prevent the immune escape of leukemic cells via regulating the Fas/FasL system and increase the sensitivity of leukemic cells to the chemotherapy drugs targeting Fas/FasL system.
10 Inhibitory effect of VEGF antisense oligonucleotides on synthesis of VEGF by Lewis lung cancer cells
2006, 13(4):281-285.
DOI: 10.3872/j.issn.1007-385X.2006.4.010
Abstract:
Objective: To study the inhibitory effect of VEGF antisense oligonucleotides (ASODN) on growth of Lewis lung cancer in C57BL/6 mice.Methods: Lewis lung cancer cells were cultured and implanted subcutaneously into 40 C57BL/6 mice, which were then divided into 4 groups: VEGF-ASODN treatment group, VEGF-SODN treatment group, BEGF-MODN treatment,and control group (normal saline). Mice in different groups were treated 24 hours after cell inoculation. The weight and volume of subcutaneous tumors was measured and the morphological changes of tumor cells was observed under microscope. VEGF protein and microvessel density were examined by immunohistochemistry.Partial tumor tissues were kept in liguid nitrogon.Results: The average tumor weights of the control, VEGF-ASODN, VEGF-SODN and VEGF-MODN groups were (7.33±0.71)g, (4.56±0.38) g, (7.59±0.32) g, and (7.62±0.39) g, respectively. The inhibition rates of tumor growth in VEGF-ASODN, VEGF-SODN and VEGF-MODN group were 43.8%, 5.5% and 3.1%, respectively. VEGF-ASPODN obviously inhibited the tumor cell growth and decelerated the tumor cell proliferation. Immunohistochemistry results showed that the expression of VEGF in ASODN group was remarkly lower than those in SODN group, MODN group and control group (P<0.05). The microvessels density (MVD) in the VEGF-ASODN, control, VEGF-SODN, and VEGF-MODN group were 8.25±2.12, 14.78±3.51, 13.71±3.62, and 12.81±2.56, respectively,with that of VEGF-ASODN remarkly lower than those of other groups（P<0.01）.Conclusion:Lewis lung cancer cells can be inhibited by the VEGF antisense oligonucleotides inoculated into tumor in the C57BL/6 mice.
Objective: To study the inhibitory effect of VEGF antisense oligonucleotides (ASODN) on growth of Lewis lung cancer in C57BL/6 mice.Methods: Lewis lung cancer cells were cultured and implanted subcutaneously into 40 C57BL/6 mice, which were then divided into 4 groups: VEGF-ASODN treatment group, VEGF-SODN treatment group, BEGF-MODN treatment,and control group (normal saline). Mice in different groups were treated 24 hours after cell inoculation. The weight and volume of subcutaneous tumors was measured and the morphological changes of tumor cells was observed under microscope. VEGF protein and microvessel density were examined by immunohistochemistry.Partial tumor tissues were kept in liguid nitrogon.Results: The average tumor weights of the control, VEGF-ASODN, VEGF-SODN and VEGF-MODN groups were (7.33±0.71)g, (4.56±0.38) g, (7.59±0.32) g, and (7.62±0.39) g, respectively. The inhibition rates of tumor growth in VEGF-ASODN, VEGF-SODN and VEGF-MODN group were 43.8%, 5.5% and 3.1%, respectively. VEGF-ASPODN obviously inhibited the tumor cell growth and decelerated the tumor cell proliferation. Immunohistochemistry results showed that the expression of VEGF in ASODN group was remarkly lower than those in SODN group, MODN group and control group (P<0.05). The microvessels density (MVD) in the VEGF-ASODN, control, VEGF-SODN, and VEGF-MODN group were 8.25±2.12, 14.78±3.51, 13.71±3.62, and 12.81±2.56, respectively,with that of VEGF-ASODN remarkly lower than those of other groups（P<0.01）.Conclusion:Lewis lung cancer cells can be inhibited by the VEGF antisense oligonucleotides inoculated into tumor in the C57BL/6 mice.
2006, 13(4):286-289.
DOI: 10.3872/j.issn.1007-385X.2006.4.011
Abstract:
Objective： To study the biological characteristics of HBsAg gene-modified DC tumor vaccine in vitro. Methods: Recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells to construct AdVHBsAg hepatocarcinoma tumor vaccine. The expression of transfected gene was detected by Western blotting. Surface molecules and phagocytosis of AdVHBsAg-DCs were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DCs was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DCs in vitro was detected by MTT assay. Results: Western blotting analysis showed that HBV surface antigen gene was expressed in transfected DCs, suggesting that the transfection was effective. AdVHBsAg-DCs up-regulated the expression of CD1a, CD11c, CD83, CD86 and HLA-DR, but lowered the phagocytosis compared with DC group (P<0.05). Autologous T cells proliferation induced by AdVHBsAg-DCs was obviously higher than DC that in control group and LacZ-DC group (P<0.05). The in vitro cytotoxicity of CTL induced by AdVHBsAg-DCs against HepG2.2.15 cells was specific. Conclusion: Hepatocarcinoma associated antigen HBsAg can be used as a key point in gene therapy of HBV related hepatoma, which provides an experimental base for immunotherapy of HBV related hepatocarcinoma mediated by DCs.
Objective： To study the biological characteristics of HBsAg gene-modified DC tumor vaccine in vitro. Methods: Recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells to construct AdVHBsAg hepatocarcinoma tumor vaccine. The expression of transfected gene was detected by Western blotting. Surface molecules and phagocytosis of AdVHBsAg-DCs were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DCs was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DCs in vitro was detected by MTT assay. Results: Western blotting analysis showed that HBV surface antigen gene was expressed in transfected DCs, suggesting that the transfection was effective. AdVHBsAg-DCs up-regulated the expression of CD1a, CD11c, CD83, CD86 and HLA-DR, but lowered the phagocytosis compared with DC group (P<0.05). Autologous T cells proliferation induced by AdVHBsAg-DCs was obviously higher than DC that in control group and LacZ-DC group (P<0.05). The in vitro cytotoxicity of CTL induced by AdVHBsAg-DCs against HepG2.2.15 cells was specific. Conclusion: Hepatocarcinoma associated antigen HBsAg can be used as a key point in gene therapy of HBV related hepatoma, which provides an experimental base for immunotherapy of HBV related hepatocarcinoma mediated by DCs.
2006, 13(4):290-295.
DOI: 10.3872/j.issn.1007-385X.2006.4.012
Abstract:
Objective: To study the relationship between serum levels of vascular endothelial growthfactor (VEGF), endostatin and pathological characteristics of patients with gastrointestinal cancer. Methods:Serum VEGF and endostatin levels were measured by enzyme-linked immunoassay in 60 patients with gastric carcinoma, 55 with hepatocellular carcinoma, 58 with colorectal carcinoma before and after surgical resection (two weeks later) and in control groups, which included 30 patients with chronic gastritis, 30 with chronic hepatitis B, 30 with adenoma of colon and 30 healthy persons. Results: The preoperative levels of VEGF and endostatin in patients with gastric carcinoma, hepatocellular carcinoma and colorectal carcinoma were significantly higher than those in patients with chronic gastritis, chronic hepatitis B, adenoma of colon and healthy persons (P<0.01). The pre-operation VEGF and endostatin levels were closely related to the grades of cell differentiation, size of the primary tumors, depth of invasion, regional lymph-node metastasis, distant metastasis and pathological stage (P<0.01), but not to tumor site and sex (P>0.05). The post-operation VEGF levels were significantly lower than that of pre-operation (P<0.01), while postoperative endostatin levels were significantly higher than that of preoperation(P<0.01). Conclusion: Elevated serum VEGF and endostatin levels in patients with gastirc, hepatocellular and colorectal carcinoma are closely correlated to the grade of cell differentiation, size of the primary tumors, invasion, metastases and pathological stages. Serum VEGF and endostatin levels may be used for evaluating the biological behavior, invasion and metastasis of gastric, hepatocellular and colorectal carcinoma preoperatively.
Objective: To study the relationship between serum levels of vascular endothelial growthfactor (VEGF), endostatin and pathological characteristics of patients with gastrointestinal cancer. Methods:Serum VEGF and endostatin levels were measured by enzyme-linked immunoassay in 60 patients with gastric carcinoma, 55 with hepatocellular carcinoma, 58 with colorectal carcinoma before and after surgical resection (two weeks later) and in control groups, which included 30 patients with chronic gastritis, 30 with chronic hepatitis B, 30 with adenoma of colon and 30 healthy persons. Results: The preoperative levels of VEGF and endostatin in patients with gastric carcinoma, hepatocellular carcinoma and colorectal carcinoma were significantly higher than those in patients with chronic gastritis, chronic hepatitis B, adenoma of colon and healthy persons (P<0.01). The pre-operation VEGF and endostatin levels were closely related to the grades of cell differentiation, size of the primary tumors, depth of invasion, regional lymph-node metastasis, distant metastasis and pathological stage (P<0.01), but not to tumor site and sex (P>0.05). The post-operation VEGF levels were significantly lower than that of pre-operation (P<0.01), while postoperative endostatin levels were significantly higher than that of preoperation(P<0.01). Conclusion: Elevated serum VEGF and endostatin levels in patients with gastirc, hepatocellular and colorectal carcinoma are closely correlated to the grade of cell differentiation, size of the primary tumors, invasion, metastases and pathological stages. Serum VEGF and endostatin levels may be used for evaluating the biological behavior, invasion and metastasis of gastric, hepatocellular and colorectal carcinoma preoperatively.
2006, 13(4):296-300.
DOI: 10.3872/j.issn.1007-385X.2006.4.013
Abstract:
Objective: To establish aneGFP-gene-marked tumor cells model in murine for long-term in vivo research and to study its preliminary application.Methods:High titer eGFP retrovirus (RV) was prepared and was used to transfect mouse leukemia cells—P388 cells. P388-eGFP clones were obtained by limited dilution method. Wild type P388 (wtP388) was used as control; DBA/2 mice were inoculated with P388-eGFP abdominally or intravenously (n=10). eGFP+ cells from abdominal cavities or bone marrow of the dyed mice were used to inoculate new mice. eGFP+ P388 cells were studied in different tissues by Flow cytometry (FCM), semi-solid culture, fluorescence microscope, light microscope and PCR, etc.Results: It was found that 80.2% P388 cells were eGFP+ 48 h after retrovirus transfection. P388-eGFP clone selection increased the eGFP+ rate to 99.2%, and the rate remained at 95.2% after 3 months′ passaging. After inoculation in abdominal cavities for (56.3±1.25) d, the eGFP+ rate of P88-eGFP cells increased to (93.3±0.50)% (n=3). eGFP gene marking had no influence on the bioactivity of P388 cells. Paraformaldehydum and liquid nitrogen both effectively protected the fluorescence signals in the tissues. Perfusion fixation method was suitable for various kinds of pathological observation approach. FCM could be used to observe eGFP+ cells in the bone marrow, liver, spleen, thymus, peripheral blood, ascitic fluid，etc. The dynamic changes of eGFP+ cells could reflect the response of tumor cells to treatment. eGFP+ cells could be used for in vitro culture and in vivo transplantation. PCR is sensitive in detecting eGFP+ tumor cells in tissues. Conclusion: The present model allows for long term, dynamic and sensitive tracing of fluorescence-labeled cells in vivo, and can be widely used in tumor and cell engineering research.
Objective: To establish aneGFP-gene-marked tumor cells model in murine for long-term in vivo research and to study its preliminary application.Methods:High titer eGFP retrovirus (RV) was prepared and was used to transfect mouse leukemia cells—P388 cells. P388-eGFP clones were obtained by limited dilution method. Wild type P388 (wtP388) was used as control; DBA/2 mice were inoculated with P388-eGFP abdominally or intravenously (n=10). eGFP+ cells from abdominal cavities or bone marrow of the dyed mice were used to inoculate new mice. eGFP+ P388 cells were studied in different tissues by Flow cytometry (FCM), semi-solid culture, fluorescence microscope, light microscope and PCR, etc.Results: It was found that 80.2% P388 cells were eGFP+ 48 h after retrovirus transfection. P388-eGFP clone selection increased the eGFP+ rate to 99.2%, and the rate remained at 95.2% after 3 months′ passaging. After inoculation in abdominal cavities for (56.3±1.25) d, the eGFP+ rate of P88-eGFP cells increased to (93.3±0.50)% (n=3). eGFP gene marking had no influence on the bioactivity of P388 cells. Paraformaldehydum and liquid nitrogen both effectively protected the fluorescence signals in the tissues. Perfusion fixation method was suitable for various kinds of pathological observation approach. FCM could be used to observe eGFP+ cells in the bone marrow, liver, spleen, thymus, peripheral blood, ascitic fluid，etc. The dynamic changes of eGFP+ cells could reflect the response of tumor cells to treatment. eGFP+ cells could be used for in vitro culture and in vivo transplantation. PCR is sensitive in detecting eGFP+ tumor cells in tissues. Conclusion: The present model allows for long term, dynamic and sensitive tracing of fluorescence-labeled cells in vivo, and can be widely used in tumor and cell engineering research.
2006, 13(4):301-302.
DOI: 10.3872/j.issn.1007-385X.2006.4.014
Abstract:
目的: 探讨几种以免疫为主的非手术治疗方法对晚期复治肺癌的疗效及其临床意义。方法: 102例晚期复治肺癌患者按主要治疗方法分单纯免疫治疗(A)、介入化疗(B)、雾化吸入免疫治疗(C)及热化疗(D)4组，观察4组的肿瘤缓解有效率、患者中位生存期及生存率。结果: A、B、C、D 4组的肿瘤缓解有效率分别为：18.8%、45.4%、23.1%、20.0%;中位生存期分别为：6.09、7.35、5.46、10.24个月，B组及D组较高; ２年以上生存率A组及D组较高，但4组间无统计差异。 结论：单纯性全身免疫疗法肿瘤缓解率较低，微创介入化疗有较高的肿瘤缓解率,但并非一定可延长生存率。局部吸入性免疫疗法及热化疗有积极意义。
目的: 探讨几种以免疫为主的非手术治疗方法对晚期复治肺癌的疗效及其临床意义。方法: 102例晚期复治肺癌患者按主要治疗方法分单纯免疫治疗(A)、介入化疗(B)、雾化吸入免疫治疗(C)及热化疗(D)4组，观察4组的肿瘤缓解有效率、患者中位生存期及生存率。结果: A、B、C、D 4组的肿瘤缓解有效率分别为：18.8%、45.4%、23.1%、20.0%;中位生存期分别为：6.09、7.35、5.46、10.24个月，B组及D组较高; ２年以上生存率A组及D组较高，但4组间无统计差异。 结论：单纯性全身免疫疗法肿瘤缓解率较低，微创介入化疗有较高的肿瘤缓解率,但并非一定可延长生存率。局部吸入性免疫疗法及热化疗有积极意义。
HAO Ming-zhi
,
CHEN Qiang
,
YE Wen-bin
,
XIAO Jing-rong
,
LIN Hai-lan
,
WU Hui
,
YU Weng-chang
,
ZHANG Kong-zhi
,
CHEN Qi-zhong
,
LIU You-xiao
,
ZHENG Wei-sheng
2006, 13(4):303-304.
DOI: 10.3872/j.issn.1007-385X.2006.4.015
Abstract:
目的: 评价肝动脉栓塞化疗联合细胞因子诱导的杀伤细胞(CIK)疗法治疗原发性肝癌的临床疗效。方法: 以生存期为观察终点指标，采用同期非随机对照方法，对2003年1月至2005年12月接受肝动脉栓塞化疗联合异体CIK细胞疗法的21例原发性肝癌患者(治疗组)与单纯肝动脉栓塞化疗46例患者(对照组)比较，观察两组的生存期差异。结果: 治疗组与对照组中位生存期分别为22个月(95％CI, 7~37)、10个月(95％CI, 8~12)。两组的半年、1年、2年生存率分别为8571%、5835%、48.62%和69.05%、32.74%、3.97%，治疗组生存期明显长于对照组（P＜0.05)。〖HT5W〗结论: 肝动脉栓塞化疗联合CIK细胞疗法较单纯肝动脉栓塞化疗有可能提高原发性肝癌患者的远期生存率。
目的: 评价肝动脉栓塞化疗联合细胞因子诱导的杀伤细胞(CIK)疗法治疗原发性肝癌的临床疗效。方法: 以生存期为观察终点指标，采用同期非随机对照方法，对2003年1月至2005年12月接受肝动脉栓塞化疗联合异体CIK细胞疗法的21例原发性肝癌患者(治疗组)与单纯肝动脉栓塞化疗46例患者(对照组)比较，观察两组的生存期差异。结果: 治疗组与对照组中位生存期分别为22个月(95％CI, 7~37)、10个月(95％CI, 8~12)。两组的半年、1年、2年生存率分别为8571%、5835%、48.62%和69.05%、32.74%、3.97%，治疗组生存期明显长于对照组（P＜0.05)。〖HT5W〗结论: 肝动脉栓塞化疗联合CIK细胞疗法较单纯肝动脉栓塞化疗有可能提高原发性肝癌患者的远期生存率。
2006, 13(4):305-307.
DOI: 10.3872/j.issn.1007-385X.2006.4.016
Abstract:
Cyr61是CCN家族成员之一，人的Cyr61基因位于染色体1q22.3，其编码的蛋白具有镶嵌型多模组结构，可与多种因子相互作用，是一种十分重要的细胞基质调节因子，在细胞的黏附、迁移与增殖以及血管生成、炎症反应和组织重构等重要生理、病理过程中发挥重要的调节作用。尤其重要的是，Cyr61参与肿瘤的发生发展过程，且在不同的肿瘤中其作用有所不同。Cyr61在乳腺癌、神经胶质瘤等肿瘤中起诱导肿瘤细胞增殖和迁移的促癌作用，而在非小细胞肺癌、子宫内膜癌等肿瘤中起到促进细胞凋亡等抑癌作用。Cyr61在不同细胞中的表达受多种信号通路的调控，可与多种整联蛋白结合激活不同的下游通路，调节Bax、Bad、p53和p21WAF1等分子的表达。作为细胞信号转导通路下游的一个重要角色，Cyr61可能成为未来肿瘤药物治疗中的重要靶点。
Cyr61是CCN家族成员之一，人的Cyr61基因位于染色体1q22.3，其编码的蛋白具有镶嵌型多模组结构，可与多种因子相互作用，是一种十分重要的细胞基质调节因子，在细胞的黏附、迁移与增殖以及血管生成、炎症反应和组织重构等重要生理、病理过程中发挥重要的调节作用。尤其重要的是，Cyr61参与肿瘤的发生发展过程，且在不同的肿瘤中其作用有所不同。Cyr61在乳腺癌、神经胶质瘤等肿瘤中起诱导肿瘤细胞增殖和迁移的促癌作用，而在非小细胞肺癌、子宫内膜癌等肿瘤中起到促进细胞凋亡等抑癌作用。Cyr61在不同细胞中的表达受多种信号通路的调控，可与多种整联蛋白结合激活不同的下游通路，调节Bax、Bad、p53和p21WAF1等分子的表达。作为细胞信号转导通路下游的一个重要角色，Cyr61可能成为未来肿瘤药物治疗中的重要靶点。
2006, 13(4):308-310.
DOI: 10.3872/j.issn.1007-385X.2006.4.017
Abstract:
血管内皮细胞生长抑制因子（vascular endothelial growth inhibitor, VEGI）是一种新型的血管内皮细胞生长抑制因子，属于 TNF超家族，是Ⅱ型跨膜蛋白。重组VEGI 不仅可以抑制内皮细胞增殖和诱导血管内皮细胞凋亡，而且可阻止新生血管生成，从而产生抗肿瘤生长的作用。VEGI作为一个内皮细胞产生的血管生成负调控因子可激活JNK、Ｐ３８ＭＡＰＡ及胱冬肽，也可激活NF-κB，从而诱导内皮细胞的凋亡。VEGI的N段部分缺失可影响其生物活性，具有重要的病理生理意义，在肿瘤生物治疗方面有很大的应用前景。
血管内皮细胞生长抑制因子（vascular endothelial growth inhibitor, VEGI）是一种新型的血管内皮细胞生长抑制因子，属于 TNF超家族，是Ⅱ型跨膜蛋白。重组VEGI 不仅可以抑制内皮细胞增殖和诱导血管内皮细胞凋亡，而且可阻止新生血管生成，从而产生抗肿瘤生长的作用。VEGI作为一个内皮细胞产生的血管生成负调控因子可激活JNK、Ｐ３８ＭＡＰＡ及胱冬肽，也可激活NF-κB，从而诱导内皮细胞的凋亡。VEGI的N段部分缺失可影响其生物活性，具有重要的病理生理意义，在肿瘤生物治疗方面有很大的应用前景。
2006, 13(4):311-314.
DOI: 10.3872/j.issn.1007-385X.2006.4.018
Abstract:
近年，NK细胞的活化性受体NKG2D已成为研究热点，作为NKG2D配体的MICA（人类MHC Ⅰ类分子链相关基因A）蛋白越来越受关注。机体内MICA以膜型和分泌型两种形式存在。膜型MICA与NKG2D相互作用在肿瘤免疫监视中起着非常重要的作用；与此相反，分泌型MICA不仅下调NKG2D受体的表达，而且下调其细胞毒活性，对免疫细胞抗肿瘤起阻碍作用，可能是肿瘤免疫逃逸的一种新的机制。因此，可以利用这些特点发挥MICA蛋白在肿瘤生物治疗中的应用价值。
近年，NK细胞的活化性受体NKG2D已成为研究热点，作为NKG2D配体的MICA（人类MHC Ⅰ类分子链相关基因A）蛋白越来越受关注。机体内MICA以膜型和分泌型两种形式存在。膜型MICA与NKG2D相互作用在肿瘤免疫监视中起着非常重要的作用；与此相反，分泌型MICA不仅下调NKG2D受体的表达，而且下调其细胞毒活性，对免疫细胞抗肿瘤起阻碍作用，可能是肿瘤免疫逃逸的一种新的机制。因此，可以利用这些特点发挥MICA蛋白在肿瘤生物治疗中的应用价值。
2006, 13(4):315-317.
DOI: 10.3872/j.issn.1007-385X.2006.4.019
Abstract:
肿瘤在人体免疫监视功能作用下仍能发生、发展、转移，表明肿瘤具有逃避机体免疫监视的功能。肿瘤自身释放的抑制因子起到重要作用。肿瘤细胞本身会释放VEGF、IL-10、TGF-β、PEG2、IL-6、趋化因子、NO等抑制因子使DC分子表面MHC分子或共刺激分子CD80/86表达改变；或是肿瘤细胞表面的标志物Fas、CD44、TAA、MHC分子、B7分子、MCRP在肿瘤表面表达改变，影响DC的抗原提呈过程，致使肿瘤无法被正常识别和杀伤。另外，T细胞应答能力的下降，在肿瘤细胞数量极少时造成漏逸，以及血清中封闭因子的存在，也会造成机体在抗肿瘤方面受到抑制。
肿瘤在人体免疫监视功能作用下仍能发生、发展、转移，表明肿瘤具有逃避机体免疫监视的功能。肿瘤自身释放的抑制因子起到重要作用。肿瘤细胞本身会释放VEGF、IL-10、TGF-β、PEG2、IL-6、趋化因子、NO等抑制因子使DC分子表面MHC分子或共刺激分子CD80/86表达改变；或是肿瘤细胞表面的标志物Fas、CD44、TAA、MHC分子、B7分子、MCRP在肿瘤表面表达改变，影响DC的抗原提呈过程，致使肿瘤无法被正常识别和杀伤。另外，T细胞应答能力的下降，在肿瘤细胞数量极少时造成漏逸，以及血清中封闭因子的存在，也会造成机体在抗肿瘤方面受到抑制。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.