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Volume 12,Issue 2,2005 Table of Contents
2005, 12(2):94-97.
DOI: 10.3872/j.issn.1007-385X.2005.2.003
Abstract:
Objective: To screen tumor antigens of nasopharyngeal carcinoma by serological proteomics technologies. Methods:The nasopharyngeal carcinoma cell line CNE-1 was used as a source of tumor cell proteins for Western blot analysis in which individual sera with special immunoreaction were selected and for immunoprecipitation analysis. Then, differential proteins are analyzed by mass spectrometry and bioinformatics methods.Results: Membrane-bound IgM was identified in the differential protein band. Conclusion: It may be a convenient, simple and sensitive way to screen tumor antigens by proteomics technologies. Membrane-bound IgM may probably be a novel antigen of nasopharyngeal carcinoma.
Objective: To screen tumor antigens of nasopharyngeal carcinoma by serological proteomics technologies. Methods:The nasopharyngeal carcinoma cell line CNE-1 was used as a source of tumor cell proteins for Western blot analysis in which individual sera with special immunoreaction were selected and for immunoprecipitation analysis. Then, differential proteins are analyzed by mass spectrometry and bioinformatics methods.Results: Membrane-bound IgM was identified in the differential protein band. Conclusion: It may be a convenient, simple and sensitive way to screen tumor antigens by proteomics technologies. Membrane-bound IgM may probably be a novel antigen of nasopharyngeal carcinoma.
2005, 12(2):98-102.
DOI: 10.3872/j.issn.1007-385X.2005.2.004
Abstract:
Objective：To construct the yeast expressive vector of rmFⅦ, in which mFⅦ was mutated to inhibit coagulation without affecting the affinity for TF, and express it in Pichia pastoris.Methods：The full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method， site-direct mutated and restriction enzyme digested as design. Cloning into pPIC9K, electroporation of Gs115, in vivo screen of multiple inserts by G418 resistance, BMGY/BMMY are used for induction and expression of rmFⅦ in pichia pastoris. These proteins were also screened for functional activity.Results：Three different rmFⅦ-pPIC9K yeast expression vectors and it′s aim protein were obtained，two kinds of proteins were found to be functional active as design. Conclusion：rmFⅦ protein can be expressed in pichia pastoris and it might facilitate the development of tumor-target molecule, and novel anti-agiogenesis drug study.
Objective：To construct the yeast expressive vector of rmFⅦ, in which mFⅦ was mutated to inhibit coagulation without affecting the affinity for TF, and express it in Pichia pastoris.Methods：The full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method， site-direct mutated and restriction enzyme digested as design. Cloning into pPIC9K, electroporation of Gs115, in vivo screen of multiple inserts by G418 resistance, BMGY/BMMY are used for induction and expression of rmFⅦ in pichia pastoris. These proteins were also screened for functional activity.Results：Three different rmFⅦ-pPIC9K yeast expression vectors and it′s aim protein were obtained，two kinds of proteins were found to be functional active as design. Conclusion：rmFⅦ protein can be expressed in pichia pastoris and it might facilitate the development of tumor-target molecule, and novel anti-agiogenesis drug study.
2005, 12(2):103-106.
DOI: 10.3872/j.issn.1007-385X.2005.2.005
Abstract:
Objective: To evaluate the antitumor activity of recombinant SEA for therapy of B16 melanoma established in C57BL/6 mice. Methods: C57BL/6 mice with melanoma were treated with the purified rSEA. The tumors were isolated and weighted. Results: Tumor growth was apparently inhibited by rSEA at high, middle, and low doses intraperitoneally, whose inhibition ratio were 79.3%, 75.6 % and 73.8% respectively. rSEA treatment in situ could inhibit tumor growth more effectively(90.6%). Further study showed that numerous CD8+ and CD4+ T cell were infiltrated in tumor tissues, which were consistent with tumor growth inhibition induced by rSEA. Conclusions: rSEA could inhibit tumor growth effectively, especially the treatment in situ. This study paves the way for tumor immunotherapy with targeted SEA.
Objective: To evaluate the antitumor activity of recombinant SEA for therapy of B16 melanoma established in C57BL/6 mice. Methods: C57BL/6 mice with melanoma were treated with the purified rSEA. The tumors were isolated and weighted. Results: Tumor growth was apparently inhibited by rSEA at high, middle, and low doses intraperitoneally, whose inhibition ratio were 79.3%, 75.6 % and 73.8% respectively. rSEA treatment in situ could inhibit tumor growth more effectively(90.6%). Further study showed that numerous CD8+ and CD4+ T cell were infiltrated in tumor tissues, which were consistent with tumor growth inhibition induced by rSEA. Conclusions: rSEA could inhibit tumor growth effectively, especially the treatment in situ. This study paves the way for tumor immunotherapy with targeted SEA.
2005, 12(2):107-110.
DOI: 10.3872/j.issn.1007-385X.2005.2.006
Abstract:
Objective: To investigate the possibility of radiolabelled immunotoxin BDI-1-MT used as bladder cancer guiding therapeutic agent, the biodistribution and metabolism of 131 I-BDI-1-MT in nude mice bearing human bladder carcinoma xenografts were studied.Methods:Monoclonal antibody BDI-1 was coupled with momordin (MT) by bifunctional agent SPDP. BDI-1 and BDI-1-MT were labeled with 131 I using ChT method. Nude mice with human bladder carcinoma BIU-87 xenografts were divided into two groups-131 I-BDI-1-MT group (10 mice) and 131 I-BDI-1 group (10 mice). In one mouse for each group, 629 MBq 131 I BDI-1-MT or 131 I BDI-1 was intravenously injected and γ imaging was performed 48 h, 72 h and 120 h postinjection. In other nine mice for each group, 0.74 MBq 131 I-BDI-1-MT or 131 I-BDI-1 was injected and biodistribution was measured at 48 h,72 h and 120 h postinjection respectively (3 mice at a time point ). The percent uptake dose per gram of tissues (%ID/g) and tumor over non-tumor radioactivity ratio (T/NT ) were calculated. Results:There was no significantly decrease in tumor uptake of 131 I-BDI-1-MT comparing with 131 I-BDI-1. The uptake of 131 I-BDI-1-MT in most of normal tissues was lower than that of 131 I-BDI-1, so T/NT values of 131 I-BDI-1-MT were higher than that of 131 I-BDI-1. The kidney uptake of131 I-BDI-1-MT was higher than that of 131 I-BDI-1. Tumor image could be seen 48 h postinjection for both 131 I-BDI-1-MT and 131 I-BDI-1 and got more clear with time lasted. But the image contrast of 131 I-BDI-1-MT was better than that of 131 I-BDI-1. There was no obvious difference in clearance rate of 131 I-BDI-1-MT and 131 I-BDI-1 in tumor. The clearance rate of 131 I-BDI-1-MT in normal tissues was significantly higher than that of 131 I-BDI-1 especially in blood. Conclusion: It demonstrated that 131 I-BDI-1-MT can effectively target to bladder cancer cells.
Objective: To investigate the possibility of radiolabelled immunotoxin BDI-1-MT used as bladder cancer guiding therapeutic agent, the biodistribution and metabolism of 131 I-BDI-1-MT in nude mice bearing human bladder carcinoma xenografts were studied.Methods:Monoclonal antibody BDI-1 was coupled with momordin (MT) by bifunctional agent SPDP. BDI-1 and BDI-1-MT were labeled with 131 I using ChT method. Nude mice with human bladder carcinoma BIU-87 xenografts were divided into two groups-131 I-BDI-1-MT group (10 mice) and 131 I-BDI-1 group (10 mice). In one mouse for each group, 629 MBq 131 I BDI-1-MT or 131 I BDI-1 was intravenously injected and γ imaging was performed 48 h, 72 h and 120 h postinjection. In other nine mice for each group, 0.74 MBq 131 I-BDI-1-MT or 131 I-BDI-1 was injected and biodistribution was measured at 48 h,72 h and 120 h postinjection respectively (3 mice at a time point ). The percent uptake dose per gram of tissues (%ID/g) and tumor over non-tumor radioactivity ratio (T/NT ) were calculated. Results:There was no significantly decrease in tumor uptake of 131 I-BDI-1-MT comparing with 131 I-BDI-1. The uptake of 131 I-BDI-1-MT in most of normal tissues was lower than that of 131 I-BDI-1, so T/NT values of 131 I-BDI-1-MT were higher than that of 131 I-BDI-1. The kidney uptake of131 I-BDI-1-MT was higher than that of 131 I-BDI-1. Tumor image could be seen 48 h postinjection for both 131 I-BDI-1-MT and 131 I-BDI-1 and got more clear with time lasted. But the image contrast of 131 I-BDI-1-MT was better than that of 131 I-BDI-1. There was no obvious difference in clearance rate of 131 I-BDI-1-MT and 131 I-BDI-1 in tumor. The clearance rate of 131 I-BDI-1-MT in normal tissues was significantly higher than that of 131 I-BDI-1 especially in blood. Conclusion: It demonstrated that 131 I-BDI-1-MT can effectively target to bladder cancer cells.
2005, 12(2):111-115.
DOI: 10.3872/j.issn.1007-385X.2005.2.007
Abstract:
Objective: To study the anti-tumor effects of suicide gene therapy using an ovarian-specific promoter and polyethylenimine(PEI) mediated transfection. Methods : (1) The pOSP1-HSVtk plasmids containing an ovarian-specific promoter were transfected using PEI into the Human ovarian carcinoma cell SKOV3, Human lung carcinoma cell NCI-H460 and Human heptocellular carcinoma cell HepG2. The cytotoxicities resulted from GCV were evaluated using the MTT assay. (2) Ovarian cancer xenograft model was established and the PEI/ pOSP1-HSVtk complex were injected around the xenograft. The expressed TK activities were estimated by monitoring the GCV concentration change using a HPLC assay. The weight of the nude mice, the tumor volumes and tumor weights were all recorded to calculate the tumor inhibition rates by weight and by volume. Histopathological analysis and TUNEL method were also performed. Results : (1) GCV was shown to be toxicity only in SKOV3 cells. (2) Compared with the control group, GCV concentrations in tumor tissues transfected pOSP1-HSVtk were shown to be significantly lower (0.05>P>0.01). The tumor volume and the tumor weight were also significantly decreased in the treated group(P<0.01). The tumor volume inhibition rate and the tumor weight inhibition rate were estimated to be 63.66% and 58.98% respectively. Histological examination revealed heavy haemorrhage and necrosis in the tumor tissues, and TUNEL confirmed substantial cell apoptosis in the treated group. Conclusion: The suicide gene therapy system using an ovarian-specific promoter by polyethylenimine mediated transfection has a targeting killing activity on human ovarian cancer.
Objective: To study the anti-tumor effects of suicide gene therapy using an ovarian-specific promoter and polyethylenimine(PEI) mediated transfection. Methods : (1) The pOSP1-HSVtk plasmids containing an ovarian-specific promoter were transfected using PEI into the Human ovarian carcinoma cell SKOV3, Human lung carcinoma cell NCI-H460 and Human heptocellular carcinoma cell HepG2. The cytotoxicities resulted from GCV were evaluated using the MTT assay. (2) Ovarian cancer xenograft model was established and the PEI/ pOSP1-HSVtk complex were injected around the xenograft. The expressed TK activities were estimated by monitoring the GCV concentration change using a HPLC assay. The weight of the nude mice, the tumor volumes and tumor weights were all recorded to calculate the tumor inhibition rates by weight and by volume. Histopathological analysis and TUNEL method were also performed. Results : (1) GCV was shown to be toxicity only in SKOV3 cells. (2) Compared with the control group, GCV concentrations in tumor tissues transfected pOSP1-HSVtk were shown to be significantly lower (0.05>P>0.01). The tumor volume and the tumor weight were also significantly decreased in the treated group(P<0.01). The tumor volume inhibition rate and the tumor weight inhibition rate were estimated to be 63.66% and 58.98% respectively. Histological examination revealed heavy haemorrhage and necrosis in the tumor tissues, and TUNEL confirmed substantial cell apoptosis in the treated group. Conclusion: The suicide gene therapy system using an ovarian-specific promoter by polyethylenimine mediated transfection has a targeting killing activity on human ovarian cancer.
2005, 12(2):116-119.
DOI: 10.3872/j.issn.1007-385X.2005.2.008
Abstract:
Objective : To construct pGL3Basic eukaryotic expression vector containing survivin promoter gene and explore the activity of this survivin promoter in Hela cells. Methods: The survivin gene promoter was amplified by polymerase chain reaction and cloned into pGL3Basic vector to construct pGL3Basic eukaryotic expression vector containing survivin gene promoter (pGL3Basic/Surp). The purified pGL3Basic/Surp was transiently transfected into HeLa cell and vessel endothelial cell line EVC304 using liposome transfection reagent and the activity of survivin gene promoter was determined by adding luciferase substrate into transfected cells 48 h later. Results: About 1 kb gene fragment was amplified by PCR method from Hela cell genomic DNA and pGL3Basic/Surp vector was constructed successfully. The activity of luciferase reporter gene was 2074.2±78.5 in Hela and 9.7±1.1 in EVC304 48 h after transfection of pGL3Basic/surp vector. Conclusion: The high specific activity of constructed survivin promoter eukaryotic expression vector might be a potential therapeutic reagent for the treatment of malignant tumor.
Objective : To construct pGL3Basic eukaryotic expression vector containing survivin promoter gene and explore the activity of this survivin promoter in Hela cells. Methods: The survivin gene promoter was amplified by polymerase chain reaction and cloned into pGL3Basic vector to construct pGL3Basic eukaryotic expression vector containing survivin gene promoter (pGL3Basic/Surp). The purified pGL3Basic/Surp was transiently transfected into HeLa cell and vessel endothelial cell line EVC304 using liposome transfection reagent and the activity of survivin gene promoter was determined by adding luciferase substrate into transfected cells 48 h later. Results: About 1 kb gene fragment was amplified by PCR method from Hela cell genomic DNA and pGL3Basic/Surp vector was constructed successfully. The activity of luciferase reporter gene was 2074.2±78.5 in Hela and 9.7±1.1 in EVC304 48 h after transfection of pGL3Basic/surp vector. Conclusion: The high specific activity of constructed survivin promoter eukaryotic expression vector might be a potential therapeutic reagent for the treatment of malignant tumor.
2005, 12(2):120-123.
DOI: 10.3872/j.issn.1007-385X.2005.2.009
Abstract:
Objective: To construct a chimeric toxin consisting of a EGF linked to PE35KDEL and evaluate the potential of the EGF-PE35KDEL to target and kill tumor cells. Methods: By using of genetic engineering techniques, a recombinant expressing plasmid pET28a-EGF-PE35KDEL was constructed, the new obtained plasmid pET28a-EGF-PE35KDEL was transformed into E.coli BL21(DE3) for proposed protein expression. EGF-PE35KDEL was purified by DEAE-Sepharose FF chromatography, cytotoxity EGF-PE35KDEL on Hela cell growth was analyzed with crystal violet staining. Results: The recombinant plasmid pet28a-EGF-PE35KDEL was constructed and expressed in E.coli. The recombinant EGF-PE35KDEL protein was expressed as a soluble protein and was up to 20% of the total protein in E. coli BL21(DE3). With the two purification procedure, the EGF-PE35KDEL is about 95% pure. The IC50 of EGF-PE35KDEL on Hela cell is 0.07 μg/ml. Conclusion: Chimeric toxin EGF-PE35KDEL had cytotoxicity to Hela cells which over expressed EGFR in vitro.
Objective: To construct a chimeric toxin consisting of a EGF linked to PE35KDEL and evaluate the potential of the EGF-PE35KDEL to target and kill tumor cells. Methods: By using of genetic engineering techniques, a recombinant expressing plasmid pET28a-EGF-PE35KDEL was constructed, the new obtained plasmid pET28a-EGF-PE35KDEL was transformed into E.coli BL21(DE3) for proposed protein expression. EGF-PE35KDEL was purified by DEAE-Sepharose FF chromatography, cytotoxity EGF-PE35KDEL on Hela cell growth was analyzed with crystal violet staining. Results: The recombinant plasmid pet28a-EGF-PE35KDEL was constructed and expressed in E.coli. The recombinant EGF-PE35KDEL protein was expressed as a soluble protein and was up to 20% of the total protein in E. coli BL21(DE3). With the two purification procedure, the EGF-PE35KDEL is about 95% pure. The IC50 of EGF-PE35KDEL on Hela cell is 0.07 μg/ml. Conclusion: Chimeric toxin EGF-PE35KDEL had cytotoxicity to Hela cells which over expressed EGFR in vitro.
LI Yue-min
,
SONG San-tai
,
JIANG Ze-fei
,
XU Jian-ming
,
ZHANG Qi
,
LI Ming-ying
,
QIAN Yan-zhen
,
CUI Zhen-fu
,
QIAN Qi-jun
2005, 12(2):124-128.
DOI: 10.3872/j.issn.1007-385X.2005.2.010
Abstract:
Objective: To evaluate the selectively oncolytic effect of conditionally replicating adenovirus CNHK500 in breast cancer. Methods: We used virus proliferation assay cell viability assay to evaluate the proliferation and cytolysis selectivity of CNHK500. And we used Western-blot to confirm the expression of adenovirus CNHK500 E1A and E1B in cancer and normal cells. Results: The CNHK500 virus proliferation ability in breast cancer cell lines is similar to that of wtAd5, better than that of ONYX-015 virus. However, CNHK500 virus replicate 1000-fold less than that of wtAd5 in normal fibroblast cell lines. CNHK500 can effectively kill breast cancer cell, while it shows attenuated cytolysis in normal fibroblast cells, with about 100-fold less than that of wtAd5. CNHK500 E1A is expressed in telomerase-positive breast cancer cells but not in telomerase-negative normal fibroblast cells. E1B protein can be detected under hypoxia condition but not in normoxia conditions. Intravenous injections of CNHK500, yielded significant tumor growth delay in the telomerase-positive breast cancer xenografts though the MCF-7 represent a very fast growing tumor cell line. Antitumor efficacy of replication-competent adenovirus CNHK500 in vivo was associated with increased dosage of CNHK500. Conclusions: The results prove that CNHK500 has highly proliferation selectivity and potent cytolysis effect on breast cancer.
Objective: To evaluate the selectively oncolytic effect of conditionally replicating adenovirus CNHK500 in breast cancer. Methods: We used virus proliferation assay cell viability assay to evaluate the proliferation and cytolysis selectivity of CNHK500. And we used Western-blot to confirm the expression of adenovirus CNHK500 E1A and E1B in cancer and normal cells. Results: The CNHK500 virus proliferation ability in breast cancer cell lines is similar to that of wtAd5, better than that of ONYX-015 virus. However, CNHK500 virus replicate 1000-fold less than that of wtAd5 in normal fibroblast cell lines. CNHK500 can effectively kill breast cancer cell, while it shows attenuated cytolysis in normal fibroblast cells, with about 100-fold less than that of wtAd5. CNHK500 E1A is expressed in telomerase-positive breast cancer cells but not in telomerase-negative normal fibroblast cells. E1B protein can be detected under hypoxia condition but not in normoxia conditions. Intravenous injections of CNHK500, yielded significant tumor growth delay in the telomerase-positive breast cancer xenografts though the MCF-7 represent a very fast growing tumor cell line. Antitumor efficacy of replication-competent adenovirus CNHK500 in vivo was associated with increased dosage of CNHK500. Conclusions: The results prove that CNHK500 has highly proliferation selectivity and potent cytolysis effect on breast cancer.
QIU Shuang
,
LU Rong
,
ZHAO Lan
,
WANG Song
,
ZHOU Chun-lei
,
ZHAO Qian
,
LI Guo-li
,
GAO Wen-yuan
,
YAO Zhi
2005, 12(2):129-133.
DOI: 10.3872/j.issn.1007-385X.2005.2.011
Abstract:
Objective: To investigate the anti-tumor effects of the tripeptide tyroserleutide (YSL) and to discuss its mechanisms by activating monocyte-macrophages. Methods: To apply human hepatocarcinoma BEL-7402 tumor transplanted in nude mice to examine the anti-tumor effects of YSL. To apply human hepatocarcinoma cell BEL-7402 to investigated the cytotoxicity of YSL against human hepatocarcinoma BEL-7402 cell line in vitro. To explore the activating effects of YSL on the peritoneal macrophage (PEMφ) functions of cytotoxicity against tumor cell lines (BEL-7402, B16-F10) in vitro and to detected the effects of YSL on the content of cytotoxicity effectors IL-1β, TNF-α and NO produced by PEMφ. Results: YSL could inhibit the growth of transplanted tumor BEL-7402 in nude mice, the inhibition rate of 160 μg/(kg·d) was 44.03%. The tumoricidal activity of YSL against BEL-7402 cell line in vitro was observed when compared with the control group ( P <0.05). YSL could activated PEMφ of nude mice and markedly enhance cytotoxicity against tumor cell lines (BEL-7402, B16-F10) when compared with the control group ( P <0.05). YSL could activated PEMφ of Balb/c mice and marked enhance cytotoxicity against tumor cell lines (BEL-7402, B16-F10) when compared with producing group ( P <0.05). YSL could stimulate the contents of the cytotoxicity effectors of IL-1β, TNF-α and NO produced by PEMφ ( P <0.05). Conclusions: YSL had inhibition functions against human hepatocarcinoma BEL-7402.YSL could increase the cytotoxicity of monocyte-macrophages and stimulate producing of the contents of the cytotoxicity effector of IL-1β, TNF-α and NO.
Objective: To investigate the anti-tumor effects of the tripeptide tyroserleutide (YSL) and to discuss its mechanisms by activating monocyte-macrophages. Methods: To apply human hepatocarcinoma BEL-7402 tumor transplanted in nude mice to examine the anti-tumor effects of YSL. To apply human hepatocarcinoma cell BEL-7402 to investigated the cytotoxicity of YSL against human hepatocarcinoma BEL-7402 cell line in vitro. To explore the activating effects of YSL on the peritoneal macrophage (PEMφ) functions of cytotoxicity against tumor cell lines (BEL-7402, B16-F10) in vitro and to detected the effects of YSL on the content of cytotoxicity effectors IL-1β, TNF-α and NO produced by PEMφ. Results: YSL could inhibit the growth of transplanted tumor BEL-7402 in nude mice, the inhibition rate of 160 μg/(kg·d) was 44.03%. The tumoricidal activity of YSL against BEL-7402 cell line in vitro was observed when compared with the control group ( P <0.05). YSL could activated PEMφ of nude mice and markedly enhance cytotoxicity against tumor cell lines (BEL-7402, B16-F10) when compared with the control group ( P <0.05). YSL could activated PEMφ of Balb/c mice and marked enhance cytotoxicity against tumor cell lines (BEL-7402, B16-F10) when compared with producing group ( P <0.05). YSL could stimulate the contents of the cytotoxicity effectors of IL-1β, TNF-α and NO produced by PEMφ ( P <0.05). Conclusions: YSL had inhibition functions against human hepatocarcinoma BEL-7402.YSL could increase the cytotoxicity of monocyte-macrophages and stimulate producing of the contents of the cytotoxicity effector of IL-1β, TNF-α and NO.
2005, 12(2):134-137.
DOI: 10.3872/j.issn.1007-385X.2005.2.012
Abstract:
Objective: To determine the parameters settings for in vivo induction and in vitro expansion of leukemia-specific CTL. Methods: C57BL/6 mice were vaccinated repeatedly with 1×10 7 DCs pulsed with 5×10 7 FBL3-lysates (FBL3-LY) every 10 days. mice were sacrificed and the spleen T cells were collected and purified. Five days after the third immunization, T cells were restimulated weekly with FBL3-HS pulsed DC(30 Gy irradiated), and low-dose IL-2 was added 10 days following. Results: Three weeks′ co-culture of splenocytes T cells from the vaccinated mice with FBL3-LY pulsed DC in the presence of low dose IL-2 resulted in more than 100-folds′ expansion of leukemia specific CTLs, which had a higher percentage of CD8 + T cell. The expanded CTLs had potent cytotoxic activity against the parental FBL3 cells（81.84±8.68%） tested in standard LDH release assay, but no cytolytic activity above background was observed against the k562 leukemia targets. Conclusion: This regime could be considered as practical alternatives to the existing clinical immunotherapy strategies.
Objective: To determine the parameters settings for in vivo induction and in vitro expansion of leukemia-specific CTL. Methods: C57BL/6 mice were vaccinated repeatedly with 1×10 7 DCs pulsed with 5×10 7 FBL3-lysates (FBL3-LY) every 10 days. mice were sacrificed and the spleen T cells were collected and purified. Five days after the third immunization, T cells were restimulated weekly with FBL3-HS pulsed DC(30 Gy irradiated), and low-dose IL-2 was added 10 days following. Results: Three weeks′ co-culture of splenocytes T cells from the vaccinated mice with FBL3-LY pulsed DC in the presence of low dose IL-2 resulted in more than 100-folds′ expansion of leukemia specific CTLs, which had a higher percentage of CD8 + T cell. The expanded CTLs had potent cytotoxic activity against the parental FBL3 cells（81.84±8.68%） tested in standard LDH release assay, but no cytolytic activity above background was observed against the k562 leukemia targets. Conclusion: This regime could be considered as practical alternatives to the existing clinical immunotherapy strategies.
LI Yong-hong
,
RAO Chun-ming
,
ZHAO Yang
,
GAO Kai
,
YUAN Li-yong
,
HAN Chun-mei
,
LI Xiang
,
WANG Jun-zhi
2005, 12(2):138-142.
DOI: 10.3872/j.issn.1007-385X.2005.2.013
Abstract:
Objective: To establish the quality control methods and requirements for recombinant adenovirus-human endostatin products. Methods: E2B region on the adenovirus vector and inserted endostatin gene were identified by PCR. The restricted enzyme digestive map of recombinant virus DNA was analysed by agarose gel eletrophoresis. The number of virus particle and infectious titer were determined by UV and TCID50 method respectively. The expression level of inserted gene was analysed by infection of human HepG2 cell. The potency of expression products was determined by its inhibition effects on the proliferation of human HM2 endothelial cell. The purity of Adv-endostatin was analysed by UV and IE-HPLC. The replication competent virus was detected by A549 cell method. Results: The PCR results of E2B region and endostatin gene on the vector were conformed to theoretics. The restricted enzyme digestive map of detected recombinant virus DNA was identical to that of the reference. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the bulk were 2.4×10 12 VP/ml, 1.53×10 11 IU/ml, and 6.4% IU/VP respectively. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the finished product were 1.0×10 12 VP/ml, 3.75×10 11 IU/ml and 3.8% IU/VP respectively. After the HepG2 cell was infected by recombinant virus for 48 hours, the concentration of endostatin in the culture was 332 ng/ml. The inhibition rate of 50 MOI recombinant adenovirus to endothelial cell was 55%. The A260nm/A280nm ratio was 1.29. The purity determined by IE-HPLC was 99.7%. There were less than one replication competent virus within 3×10 10 VP recombinant adenovirus products. Other items complied with its corresponding requirements. Conclusion: The methods and requirements had been established for quality control of Adv-human endostatin.
Objective: To establish the quality control methods and requirements for recombinant adenovirus-human endostatin products. Methods: E2B region on the adenovirus vector and inserted endostatin gene were identified by PCR. The restricted enzyme digestive map of recombinant virus DNA was analysed by agarose gel eletrophoresis. The number of virus particle and infectious titer were determined by UV and TCID50 method respectively. The expression level of inserted gene was analysed by infection of human HepG2 cell. The potency of expression products was determined by its inhibition effects on the proliferation of human HM2 endothelial cell. The purity of Adv-endostatin was analysed by UV and IE-HPLC. The replication competent virus was detected by A549 cell method. Results: The PCR results of E2B region and endostatin gene on the vector were conformed to theoretics. The restricted enzyme digestive map of detected recombinant virus DNA was identical to that of the reference. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the bulk were 2.4×10 12 VP/ml, 1.53×10 11 IU/ml, and 6.4% IU/VP respectively. The number of virus particle, the infectious titer and the ratio of infectious titer to virus particle for the finished product were 1.0×10 12 VP/ml, 3.75×10 11 IU/ml and 3.8% IU/VP respectively. After the HepG2 cell was infected by recombinant virus for 48 hours, the concentration of endostatin in the culture was 332 ng/ml. The inhibition rate of 50 MOI recombinant adenovirus to endothelial cell was 55%. The A260nm/A280nm ratio was 1.29. The purity determined by IE-HPLC was 99.7%. There were less than one replication competent virus within 3×10 10 VP recombinant adenovirus products. Other items complied with its corresponding requirements. Conclusion: The methods and requirements had been established for quality control of Adv-human endostatin.
2005, 12(2):143-147.
DOI: 10.3872/j.issn.1007-385X.2005.2.014
Abstract:
Objective: To investigate the inhibitory effect of telomerase activity by chimeric oligonucleotide.Methods: Here various oligonucleotides were designed and synthesized. Inactivity of the telomerase activity inhibitors was observed with HL-60 cell-lysates in vitro and with U-87 clell in vivo. Results: Both phosphorothioate-modified oligonuclotides (PS-ODNs) and oligonucleotides complementary with telomerase RNA template inhibited telomerase activity. And application of the tow ODNs together increased the inhibition activity in upfold. the various modification extensional oligonucleotides complementary with telomerase RNA template at 3′-end of the PS-ODNs enabled the chimeric oligonucleotides to increase their inhibition efficiency. Conclusion: These cODNs emerged as powerful inhibitors of human telomerase detected so for and might be promising candidates to investigate the effect of permanent of inhibition of telomerse on tumor growth.
Objective: To investigate the inhibitory effect of telomerase activity by chimeric oligonucleotide.Methods: Here various oligonucleotides were designed and synthesized. Inactivity of the telomerase activity inhibitors was observed with HL-60 cell-lysates in vitro and with U-87 clell in vivo. Results: Both phosphorothioate-modified oligonuclotides (PS-ODNs) and oligonucleotides complementary with telomerase RNA template inhibited telomerase activity. And application of the tow ODNs together increased the inhibition activity in upfold. the various modification extensional oligonucleotides complementary with telomerase RNA template at 3′-end of the PS-ODNs enabled the chimeric oligonucleotides to increase their inhibition efficiency. Conclusion: These cODNs emerged as powerful inhibitors of human telomerase detected so for and might be promising candidates to investigate the effect of permanent of inhibition of telomerse on tumor growth.
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.