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Volume 13,Issue 3,2006 Table of Contents
2006, 13(3):159-161.
DOI: 10.3872/j.issn.1007-385X.2006.3.001
Abstract:
CAO Li-min
,
WANG Wei-yu
,
ZHAO Xiao-rong
,
YE Qing
,
LEI Ping
,
LIU Jing
,
DAI Wei
,
ZHU Yin-chang
,
SI Jin
,
SHEN Guan-xin
2006, 13(3):162-166.
DOI: 10.3872/j.issn.1007-385X.2006.3.002
Abstract:
Objective： To explore the killing effect of the fusion protein (HSV1-TK fused with an 11-amino-acid peptide of the basic domain of the HIV-1 Tat protein, Tat11) on hepatoma cell line HepG2. Methods：Two single oligonucleotide chains encoding HIV-Tat47-57（Tat11）were synthesized and were used for producing double strand oligonucleotide through introducing BamHⅠ and HindⅢ and annealing. 15% non-denaturing polyacrylamide gel electrophoresis was used for analysis of the annealing result. The full length cDNA encoding HSV1-TK was amplified from r-pAs16Dr by PCR. Then the 2 fragments were sub-cloned into a preukaryotic expression vector (pET-32c). A single colony of E. coli BL21 containing the plasmid pET-32c-Tat11-TK was innoculated LB broth, diluted 1/100 into 1 000 ml LB broth, and was then treated with 1 mmol/L IPTG. The recombinant Tat11-TK was purified with Ni2+ chelating HiTrap HP column and its intercellular translocation ability was evaluated by immnunohistochemistry and Western blot. Results： The recombinant Tat11-TK protein showed bands at about 60 700, which was in accordance with the theoretical value of the fusion protein, also proved the presence of inclusion body. The result of immnunohistochemistry showed that Tat11-TK could bind to the cell surface and Western blot showed that it could also effectively enter into the HepG2. It was also found that HepG2 was more sensitive to GCV（150 μmol/L） in the presence of Tat11-TK. Conclusion： The fused protein Tat11-TK can be highly expressed in a preukaryotic system and has potent ability for membrane perforation and enzyme trafficking.
Objective： To explore the killing effect of the fusion protein (HSV1-TK fused with an 11-amino-acid peptide of the basic domain of the HIV-1 Tat protein, Tat11) on hepatoma cell line HepG2. Methods：Two single oligonucleotide chains encoding HIV-Tat47-57（Tat11）were synthesized and were used for producing double strand oligonucleotide through introducing BamHⅠ and HindⅢ and annealing. 15% non-denaturing polyacrylamide gel electrophoresis was used for analysis of the annealing result. The full length cDNA encoding HSV1-TK was amplified from r-pAs16Dr by PCR. Then the 2 fragments were sub-cloned into a preukaryotic expression vector (pET-32c). A single colony of E. coli BL21 containing the plasmid pET-32c-Tat11-TK was innoculated LB broth, diluted 1/100 into 1 000 ml LB broth, and was then treated with 1 mmol/L IPTG. The recombinant Tat11-TK was purified with Ni2+ chelating HiTrap HP column and its intercellular translocation ability was evaluated by immnunohistochemistry and Western blot. Results： The recombinant Tat11-TK protein showed bands at about 60 700, which was in accordance with the theoretical value of the fusion protein, also proved the presence of inclusion body. The result of immnunohistochemistry showed that Tat11-TK could bind to the cell surface and Western blot showed that it could also effectively enter into the HepG2. It was also found that HepG2 was more sensitive to GCV（150 μmol/L） in the presence of Tat11-TK. Conclusion： The fused protein Tat11-TK can be highly expressed in a preukaryotic system and has potent ability for membrane perforation and enzyme trafficking.
2006, 13(3):167-170.
DOI: 10.3872/j.issn.1007-385X.2006.3.003
Abstract:
Objective: To investigate the in vitro efficiency of two kinds of polylatic acid (PLA) immunomicrospheres: M1（hAFP158~166）and M2（hAFP218~226） in specific inducement of CTL and the cytotoxicity of CTL against hepatocellular carcinoma cells. Methods: Pripheral blood monocytes (PBMC) of HLA-A2+ healthy volunteers stimulated in vitro by peptide-loading microspheres were taken as effectors. The experiments were divided into three groups: control group, hAFP+ tumor cells group, and hAFP- tumor cells group. The specific cytolysis of target cells was assessed by 51Cr-release assay. Results: Both M1 and M2 induced proliferation of HLA-A2+ PBMC, forming visible clones. The effector cells induced by M1 and M2 both had an cytolysis rate over 75% for hAFP+ tumor cells, which was significantly higher than that for hAFP- tumor cells (P<0.01). But there was no significant difference in cytolysis rate concerning the two kinds of microspheres (P>0.05). Conclusion: The two kinds of microspheres can both induce specific CTL in vitro, which can effectively kill hAFP+ tumor cells.
Objective: To investigate the in vitro efficiency of two kinds of polylatic acid (PLA) immunomicrospheres: M1（hAFP158~166）and M2（hAFP218~226） in specific inducement of CTL and the cytotoxicity of CTL against hepatocellular carcinoma cells. Methods: Pripheral blood monocytes (PBMC) of HLA-A2+ healthy volunteers stimulated in vitro by peptide-loading microspheres were taken as effectors. The experiments were divided into three groups: control group, hAFP+ tumor cells group, and hAFP- tumor cells group. The specific cytolysis of target cells was assessed by 51Cr-release assay. Results: Both M1 and M2 induced proliferation of HLA-A2+ PBMC, forming visible clones. The effector cells induced by M1 and M2 both had an cytolysis rate over 75% for hAFP+ tumor cells, which was significantly higher than that for hAFP- tumor cells (P<0.01). But there was no significant difference in cytolysis rate concerning the two kinds of microspheres (P>0.05). Conclusion: The two kinds of microspheres can both induce specific CTL in vitro, which can effectively kill hAFP+ tumor cells.
2006, 13(3):171-175.
DOI: 10.3872/j.issn.1007-385X.2006.3.004
Abstract:
Objective: To investigate the inhibitory effects of PTEN gene transfection combined with L-OHP on human cholangiocarcinoma cell line, QBC939, providing a new method for gene therapy of human biliary duct carcinoma. Methods: A eukaryotic expression vector containing PTEN gene was transfected into human QBC939 cells under mediation of lipofectamine and positive cell clones were selected and amplified. Expression of PTEN gene was detected by immunohistochemistry. MTT test was used to determine the in vitro activity of cells, electron microscope was applied to observe cell ultrastructure, and flow cytometry was used for determining the cell cycle and apoptosis. In vitro test was used to study the invasive ability of cells before and after treatment. Results: After transfected with PTEN gene, QBC939 cells had a higher expression of PTEN gene (P<0.05), a decreased activity (P<0.05), an arrest of G1-S phase, and an increased apoptotic rate (P<0.01). Electron microscope showed that maturity of cells and increased well-differentiated mitochondria. The aggressiveness of the cells was obviously inhibited (P<0.05). Conclusion: PTEN gene transfection combined with L-OHP has obvious inhibitory effect on cholangiocarcinoma cell line in vitro.
Objective: To investigate the inhibitory effects of PTEN gene transfection combined with L-OHP on human cholangiocarcinoma cell line, QBC939, providing a new method for gene therapy of human biliary duct carcinoma. Methods: A eukaryotic expression vector containing PTEN gene was transfected into human QBC939 cells under mediation of lipofectamine and positive cell clones were selected and amplified. Expression of PTEN gene was detected by immunohistochemistry. MTT test was used to determine the in vitro activity of cells, electron microscope was applied to observe cell ultrastructure, and flow cytometry was used for determining the cell cycle and apoptosis. In vitro test was used to study the invasive ability of cells before and after treatment. Results: After transfected with PTEN gene, QBC939 cells had a higher expression of PTEN gene (P<0.05), a decreased activity (P<0.05), an arrest of G1-S phase, and an increased apoptotic rate (P<0.01). Electron microscope showed that maturity of cells and increased well-differentiated mitochondria. The aggressiveness of the cells was obviously inhibited (P<0.05). Conclusion: PTEN gene transfection combined with L-OHP has obvious inhibitory effect on cholangiocarcinoma cell line in vitro.
2006, 13(3):176-180.
DOI: 10.3872/j.issn.1007-385X.2006.3.005
Abstract:
Objective: To investigate the efficiency of carbon nanotube(CNT)-PAMAM mediated entrance of anti-survivin oligonucleotide into HepG2 cells, and its effects on the proliferation of HepG2 cells. Methods: CNT-PAMAM-anti-survivin oligonucleotide compounds were prepared and characterized by AFM and 1% agarose gel electrophoresis analysis. TEM was used to observe the distribution of CNT-PAMAM-ASODN compounds in HepG2 cells. CNT-PAMAM-ASODN compounds were added into the medium and co-cultured with HepG2 cells for 24 h, 48 h,72 h, and 96 h at 37℃, 5% CO2. MTT method was used to detect the effects of ASODN and CNT-PAMAM-ASODN on the proliferation of HepG2 cells. Results: CNT-PAMAM-ASODN compounds were successfully synthesized via AFM and agarose gel electrophoresis. TEM showed that the compounds were located in the cytoplasm. When CNT-PAMAM-ASODN (1.0 μmol/L) and ASODN (1.0 μmol/L) were used for a 48 h culture, the inhibitory rates of HepG2 cells were (45.97±4.28) % for CNT-PAMAM-ASODN compounds group, (9.33±0.85)% for ASODN group, and (6.37±0.69)% for CNT-PAMAM group. CNT-PAMAM-ASODN compounds at 1.5 μmol/L inhibited HepG2 cells by (70.22±7.25)%, and the inhibitory effects were in a time- and concentration-dependent manner. There was statistical difference between experiment group and control group (P<0.01). Conclusion: CNT-PAMAM compounds may serve as a gene delivery vector with high efficiency, which can bring survivin ASODN into HepG2 cells, inhibiting HepG2 cell proliferation and markedly enhancing the inhibitory effects of survivin against HepG2 cells.
Objective: To investigate the efficiency of carbon nanotube(CNT)-PAMAM mediated entrance of anti-survivin oligonucleotide into HepG2 cells, and its effects on the proliferation of HepG2 cells. Methods: CNT-PAMAM-anti-survivin oligonucleotide compounds were prepared and characterized by AFM and 1% agarose gel electrophoresis analysis. TEM was used to observe the distribution of CNT-PAMAM-ASODN compounds in HepG2 cells. CNT-PAMAM-ASODN compounds were added into the medium and co-cultured with HepG2 cells for 24 h, 48 h,72 h, and 96 h at 37℃, 5% CO2. MTT method was used to detect the effects of ASODN and CNT-PAMAM-ASODN on the proliferation of HepG2 cells. Results: CNT-PAMAM-ASODN compounds were successfully synthesized via AFM and agarose gel electrophoresis. TEM showed that the compounds were located in the cytoplasm. When CNT-PAMAM-ASODN (1.0 μmol/L) and ASODN (1.0 μmol/L) were used for a 48 h culture, the inhibitory rates of HepG2 cells were (45.97±4.28) % for CNT-PAMAM-ASODN compounds group, (9.33±0.85)% for ASODN group, and (6.37±0.69)% for CNT-PAMAM group. CNT-PAMAM-ASODN compounds at 1.5 μmol/L inhibited HepG2 cells by (70.22±7.25)%, and the inhibitory effects were in a time- and concentration-dependent manner. There was statistical difference between experiment group and control group (P<0.01). Conclusion: CNT-PAMAM compounds may serve as a gene delivery vector with high efficiency, which can bring survivin ASODN into HepG2 cells, inhibiting HepG2 cell proliferation and markedly enhancing the inhibitory effects of survivin against HepG2 cells.
6 Effect of blocking BRCAA1 gene with siRNA on proliferation of MCF-7 cells and expression of Rb gene
YANG Hao
,
CUI Da-xiang
,
LI Qing
,
HUANG Tuo
,
GAO Feng
,
HE Rong
,
PAN Bi-feng
,
SHAO Jun
,
YOU Xiao-gang
,
LIU Feng-tao
2006, 13(3):181-184.
DOI: 10.3872/j.issn.1007-385X.2006.3.006
Abstract:
Objective: To investigate the effect of blocking BRCAA1 gene expression with siRNA on the proliferation of tumor cell line MCF-7 and Rb gene expression. Methods: RNAi was employed to specifically knock down BRCAA1 expression. MCF-7 cells were transfected with complexes constructed with lipids and chemically synthesized Pre-designed anti-BRCAA1 siRNAs. The total RNA was isolated and reversely transcribed after 48 h. The expressions of BRCAA1 and Rb mRNA were determined by Real-Time PCR. Results: Compared with negative control, transfected MCF-7 cells had a 42.3% decrease in expression of BRCAA1 mRNA and an 11.1% increase in Rb mRNA expression. The inhibitory rate of MCF-7 cells proliferation was (81.6±61)%. Conclusion: There may be some antagonistic effect between BRCAA1 gene and Rb gene in proliferation of tumor cells, which provides a potential target for anti-tumor gene therapy.
Objective: To investigate the effect of blocking BRCAA1 gene expression with siRNA on the proliferation of tumor cell line MCF-7 and Rb gene expression. Methods: RNAi was employed to specifically knock down BRCAA1 expression. MCF-7 cells were transfected with complexes constructed with lipids and chemically synthesized Pre-designed anti-BRCAA1 siRNAs. The total RNA was isolated and reversely transcribed after 48 h. The expressions of BRCAA1 and Rb mRNA were determined by Real-Time PCR. Results: Compared with negative control, transfected MCF-7 cells had a 42.3% decrease in expression of BRCAA1 mRNA and an 11.1% increase in Rb mRNA expression. The inhibitory rate of MCF-7 cells proliferation was (81.6±61)%. Conclusion: There may be some antagonistic effect between BRCAA1 gene and Rb gene in proliferation of tumor cells, which provides a potential target for anti-tumor gene therapy.
7 Sodium butyrate induces apoptosis and regulates p 53 target genes in HT-29 colorectal cancer cells
LIU Cheng-xia
,
ZHANG Shang-zhong
,
ZHANG Xiao-wei
,
HUANG Li-hua
,
LI Tie-jun
,
ZHANG Jing
,
WANG Bing
2006, 13(3):185-190.
DOI: 10.3872/j.issn.1007-385X.2006.3.007
Abstract:
Objective: To investigate the regulatory effects of sodium butyrate onp53 target genes (p21waf1, bax,and gadd45) in HT-29 colorectal cancer cells and the related mechanisms. Methods: HT-29 cells were cultured in the absence or presence of sodium butyrate. The cell proliferation and cell cycle were studied by MTT and FCM, respectively. Apoptosis was assessed by observing cell morphology, percentage of sub-G1 cells and AnnexinV-FITC. The effects of sodium butyrate on transcription of p21waf1, bax and gadd45 were analyzed by RT-PCR and Western blot. Results: Sodium butyrate inhibited proliferation and induced apoptosis of HT-29 cells in a time- and dose-dependent manner, and it blocked HT-29 cell at G1 phase. Sodium butyrate stimulated p21waf1 and bax expression both at mRNA and protein level in HT-29 cells, but had little effect on the transcription of gadd45. Conclusion: Sodium butyrate can inhibit proliferation and induce apoptosis of HT-29 cells, which might be through up-regulating p21waf1 and -bax-expression both at mRNA and protein levels.
Objective: To investigate the regulatory effects of sodium butyrate onp53 target genes (p21waf1, bax,and gadd45) in HT-29 colorectal cancer cells and the related mechanisms. Methods: HT-29 cells were cultured in the absence or presence of sodium butyrate. The cell proliferation and cell cycle were studied by MTT and FCM, respectively. Apoptosis was assessed by observing cell morphology, percentage of sub-G1 cells and AnnexinV-FITC. The effects of sodium butyrate on transcription of p21waf1, bax and gadd45 were analyzed by RT-PCR and Western blot. Results: Sodium butyrate inhibited proliferation and induced apoptosis of HT-29 cells in a time- and dose-dependent manner, and it blocked HT-29 cell at G1 phase. Sodium butyrate stimulated p21waf1 and bax expression both at mRNA and protein level in HT-29 cells, but had little effect on the transcription of gadd45. Conclusion: Sodium butyrate can inhibit proliferation and induce apoptosis of HT-29 cells, which might be through up-regulating p21waf1 and -bax-expression both at mRNA and protein levels.
ZHANG Lang-hui
,
LIU Yong-jun
,
Lü Lu-lu
,
WANG Ai-ping
,
XU Zhen-shu
,
ZHU Xiong-peng
,
CHEN Zhi-zhe
,
HAN Zhong-chao
2006, 13(3):191-195.
DOI: 10.3872/j.issn.1007-385X.2006.3.008
Abstract:
Objective: To investigate the effects of human umbilical cord derived mesenchymal stem cells (hUC-MSCs) on the activation and proliferation of allogenic umbilical core blood T lymphocytes, so as to study the immunomodulatory capacity of hUC-MSC. Methods: hUC-MSCs were isolated, culture-expanded from human umbilical cord after enzyme digestion, and the major histocompatibility (MHC) phenotype features of hUC-MSC were detected. The experiment was divided into 3 groups: (1) Negative group: umbilical cord blood MNCs were cultured alone; (2) Control group: MNCs were cultured with nonspecific mitogenic stimuli; (3) Experimental group: MNCs were co-cultured with hUC-MSC and nonspecific mitogenic stimuli. FCM technique was used to detect the CD69 expression, an indicator of T-cell early activation, on T cells and CD4+, CD8+ subsets after 8 hours' co-culture. T-lymphocyte proliferation in each group was detected by MTT after 5 days' co-culture. Results: We successfully established a simple method to isolate and culture-expand abundant hUC-MSCs from human umbilical cord. The immunophenotypic analysis showed that hUC-MSCs expressed no HLA class Ⅱ and less HLA class Ⅰ. hUC-MSC inhibited CD69 expression in PHA and PMA activated T cells from (22.6±5.2)% to (7.8±3.5)%（P<0.01）after 8 hours' co-culture, which influenced both CD4+ and CD8+ T-cell subsets. MTT showed that hUC-MSC dose-dependently inhibited PHA-induced T-lymphocyte proliferation. Conclusion: hUC-MSC has a dose-dependent inhibitory effect on nonspecific mitogenic stimuli triggered activation and proliferation of allogeneic umbilical cord blood T lymphocytes, and the effect is not selective.
Objective: To investigate the effects of human umbilical cord derived mesenchymal stem cells (hUC-MSCs) on the activation and proliferation of allogenic umbilical core blood T lymphocytes, so as to study the immunomodulatory capacity of hUC-MSC. Methods: hUC-MSCs were isolated, culture-expanded from human umbilical cord after enzyme digestion, and the major histocompatibility (MHC) phenotype features of hUC-MSC were detected. The experiment was divided into 3 groups: (1) Negative group: umbilical cord blood MNCs were cultured alone; (2) Control group: MNCs were cultured with nonspecific mitogenic stimuli; (3) Experimental group: MNCs were co-cultured with hUC-MSC and nonspecific mitogenic stimuli. FCM technique was used to detect the CD69 expression, an indicator of T-cell early activation, on T cells and CD4+, CD8+ subsets after 8 hours' co-culture. T-lymphocyte proliferation in each group was detected by MTT after 5 days' co-culture. Results: We successfully established a simple method to isolate and culture-expand abundant hUC-MSCs from human umbilical cord. The immunophenotypic analysis showed that hUC-MSCs expressed no HLA class Ⅱ and less HLA class Ⅰ. hUC-MSC inhibited CD69 expression in PHA and PMA activated T cells from (22.6±5.2)% to (7.8±3.5)%（P<0.01）after 8 hours' co-culture, which influenced both CD4+ and CD8+ T-cell subsets. MTT showed that hUC-MSC dose-dependently inhibited PHA-induced T-lymphocyte proliferation. Conclusion: hUC-MSC has a dose-dependent inhibitory effect on nonspecific mitogenic stimuli triggered activation and proliferation of allogeneic umbilical cord blood T lymphocytes, and the effect is not selective.
2006, 13(3):196-199.
DOI: 10.3872/j.issn.1007-385X.2006.3.009
Abstract:
Objective: To study the inhibitory effects of magnetic poly D,L-lactide-co-glycolide phenylarsine oxide nanoparticles (M-PLGA-PAO-NS) on the growth of human hepatic carcinoma cells．Methods： The magnetic polyD,L-lactide-co-glycolide phenylarsine oxide nano-particles were prepared by emulsion-evaporation process. The growth inhibition of tumor cell was studied by the microscope observation and MTT method, and cell apoptosis was observed by FCM．Results: The prepared M-PLGA-PAO-NS had a regular spherical surface and a mean diameter of 290 nm. The magnetic nano-particles effectively inhibited the growth of carcinoma cells (SMMC-7721) in a time- and concentration-dependent manner \[e.g. inhibitory rate of 1.0 μmol/L nano-particles (4.6±0.9)% for 24 h versus (11.4±1.2)% for 48 h \].FCM assay indicated that most of the cells were arrested at S+G2/M phase and there was apoptotic peak . Conclusion： The prepared M-PLGA-PAO-NS has high anti-hepatic carcinoma activity against SMMC-7721, and cells in the G0/G1 phase may be the target for drug action.
Objective: To study the inhibitory effects of magnetic poly D,L-lactide-co-glycolide phenylarsine oxide nanoparticles (M-PLGA-PAO-NS) on the growth of human hepatic carcinoma cells．Methods： The magnetic polyD,L-lactide-co-glycolide phenylarsine oxide nano-particles were prepared by emulsion-evaporation process. The growth inhibition of tumor cell was studied by the microscope observation and MTT method, and cell apoptosis was observed by FCM．Results: The prepared M-PLGA-PAO-NS had a regular spherical surface and a mean diameter of 290 nm. The magnetic nano-particles effectively inhibited the growth of carcinoma cells (SMMC-7721) in a time- and concentration-dependent manner \[e.g. inhibitory rate of 1.0 μmol/L nano-particles (4.6±0.9)% for 24 h versus (11.4±1.2)% for 48 h \].FCM assay indicated that most of the cells were arrested at S+G2/M phase and there was apoptotic peak . Conclusion： The prepared M-PLGA-PAO-NS has high anti-hepatic carcinoma activity against SMMC-7721, and cells in the G0/G1 phase may be the target for drug action.
2006, 13(3):200-204.
DOI: 10.3872/j.issn.1007-385X.2006.3.010
Abstract:
Objective: To investigate the effects of human IL-24 on phenotype and antigen presenting function of human monocyte-derived dendritic cells in vitro. Methods: Western blot was used to determine the level of IL-24 in the DC culture supernatant.Semi-quantitative RT-PCR was used to analyze the expression of IL-24 receptor and chemokine receptors in DCs at different developmental stages. Fluorescence-activated cell sorting (FACS) analysis was used to assess the expression of MHC class Ⅱ molecule, CD80 and CD86 on DCs surface in the presence of IL-24. Allogenic mixed lymphocyte reaction was employed to analyze the effect of IL-24 on the antigen presenting function of DCs. Results: We found that LPS induced DCs to secrete endogenous IL-24. Meanwhile, LPS also up-regulated the expression of IL-22R1, an IL-24 receptor subunit, on DCs. Supplement of IL-24 to DCs culture medium significantly augmented the expression of MHCⅡ, CD80, and CD86 on DCs surface, and promoted the antigen presenting function of DCs to T cells. Conclusion: IL-24 can promote the activation and maturation of DCs, increasing the antigen presenting function of DCs.
Objective: To investigate the effects of human IL-24 on phenotype and antigen presenting function of human monocyte-derived dendritic cells in vitro. Methods: Western blot was used to determine the level of IL-24 in the DC culture supernatant.Semi-quantitative RT-PCR was used to analyze the expression of IL-24 receptor and chemokine receptors in DCs at different developmental stages. Fluorescence-activated cell sorting (FACS) analysis was used to assess the expression of MHC class Ⅱ molecule, CD80 and CD86 on DCs surface in the presence of IL-24. Allogenic mixed lymphocyte reaction was employed to analyze the effect of IL-24 on the antigen presenting function of DCs. Results: We found that LPS induced DCs to secrete endogenous IL-24. Meanwhile, LPS also up-regulated the expression of IL-22R1, an IL-24 receptor subunit, on DCs. Supplement of IL-24 to DCs culture medium significantly augmented the expression of MHCⅡ, CD80, and CD86 on DCs surface, and promoted the antigen presenting function of DCs to T cells. Conclusion: IL-24 can promote the activation and maturation of DCs, increasing the antigen presenting function of DCs.
2006, 13(3):205-208.
DOI: 10.3872/j.issn.1007-385X.2006.3.011
Abstract:
Objective: To investigate the in vitro the anti-prostate cancer effect of a novel polypeptide, APP216, so as to provide a basis for development of new drug for treatment of prostate cancer. Methods: The polypeptide drug included the amino sequences of BH3, K237 and DG2 domain and the peptide that could be digested by PSA. The anti-prostate cancer effects of the polypeptide prodrug on prostate cancer cell line LNCaP, 22RV1 (secreting PSA) and PC3, DU145 (secreting no PSA) were determined by MTT test and Hoechst 33258 staining. Results: MTT test revealed that the surviving rates of LNCaP and 22PV1 cells were respectively 22% and 34% 48 h after APP216 (270 μg/ml) treatment, and 9.8% and 8.2% 72 h after APP216 (270 μg/ml) treatment. The surviving rates of PC3 and DU145 cells were respectively 90% and 95% 48 h after APP216 (270 μg/ml) treatment, and 87% and 92% 72 h after APP216 (270 μg/ml) treatment. Hoechst 33258 staining showed the typical features of cell apoptosis: cell shrinkage, chromatin condensation and hypodiploid genomic DNA content in LNCaP and PC3m cells. Flow cytometry showed an apotosis rate of 36.26% 48 h after APP216 (270 μg/ml) treatment in LNCaP cells, and of 1.63% after 48 h APP216 (270 μg/ml) treatment in PC3m cells.Conclusion: APP216 has a satisfactory in vitro cytotoxicity on human PSA-secreting prostate cancer cells and can induce tumor cell apoptosis, but not on non-PSA secreting prostate cancer cells, indicating APP216 polypeptide can be specifically digested by endonuclease enzyme. Meanwhile, BH3 domain can be transferred into cells and induce apoptosis through HIV-TAT.
Objective: To investigate the in vitro the anti-prostate cancer effect of a novel polypeptide, APP216, so as to provide a basis for development of new drug for treatment of prostate cancer. Methods: The polypeptide drug included the amino sequences of BH3, K237 and DG2 domain and the peptide that could be digested by PSA. The anti-prostate cancer effects of the polypeptide prodrug on prostate cancer cell line LNCaP, 22RV1 (secreting PSA) and PC3, DU145 (secreting no PSA) were determined by MTT test and Hoechst 33258 staining. Results: MTT test revealed that the surviving rates of LNCaP and 22PV1 cells were respectively 22% and 34% 48 h after APP216 (270 μg/ml) treatment, and 9.8% and 8.2% 72 h after APP216 (270 μg/ml) treatment. The surviving rates of PC3 and DU145 cells were respectively 90% and 95% 48 h after APP216 (270 μg/ml) treatment, and 87% and 92% 72 h after APP216 (270 μg/ml) treatment. Hoechst 33258 staining showed the typical features of cell apoptosis: cell shrinkage, chromatin condensation and hypodiploid genomic DNA content in LNCaP and PC3m cells. Flow cytometry showed an apotosis rate of 36.26% 48 h after APP216 (270 μg/ml) treatment in LNCaP cells, and of 1.63% after 48 h APP216 (270 μg/ml) treatment in PC3m cells.Conclusion: APP216 has a satisfactory in vitro cytotoxicity on human PSA-secreting prostate cancer cells and can induce tumor cell apoptosis, but not on non-PSA secreting prostate cancer cells, indicating APP216 polypeptide can be specifically digested by endonuclease enzyme. Meanwhile, BH3 domain can be transferred into cells and induce apoptosis through HIV-TAT.
REN Peng-kang
,
WEI Fang
,
LI Hui-ming
,
WANG Feng
,
ZHANG Ju-feng
,
CHEN Xia-fang
,
LI Zong-hai
,
HUANG Qian
2006, 13(3):209-213.
DOI: 10.3872/j.issn.1007-385X.2006.3.012
Abstract:
Objective: To investigate the relationship between adenovirus type 5 infection efficiency and expression of Coxsackie virus adenovirus receptor (CAR) and integrin in different tumor cell lines, providing a basis for further study of adenovirus gene therapy. Methods: The expression levels of CAR in different cell lines were examined by flow cytometry. The cells were infected with Ad5-CMV-GFP and Ad5-CMV-luc carrying report genes, tested the infection efficiency and determined their expression levels by flow cytometry and Luciferase Assay. The variation of adenovirus infection efficiency was evaluated and compared when the CAR expression was enhanced on cell surface with plasmid expressing CAR or blocked by antibodies of CAR and integrin. Results: The highest CAR expression was found in SMMC-7721 and A549 cell lines, the lowest in K562 cells. The results of fluorescence microscope and flow cytometry suggested that Ad5 had the highest efficiency in infecting SMMC-7721 and A549 cell lines, and the lowest in infecting K562 cells. Conclusion：The infection efficiency of adenovirus is determined by the levels of CAR and integrin on cancer cells.
Objective: To investigate the relationship between adenovirus type 5 infection efficiency and expression of Coxsackie virus adenovirus receptor (CAR) and integrin in different tumor cell lines, providing a basis for further study of adenovirus gene therapy. Methods: The expression levels of CAR in different cell lines were examined by flow cytometry. The cells were infected with Ad5-CMV-GFP and Ad5-CMV-luc carrying report genes, tested the infection efficiency and determined their expression levels by flow cytometry and Luciferase Assay. The variation of adenovirus infection efficiency was evaluated and compared when the CAR expression was enhanced on cell surface with plasmid expressing CAR or blocked by antibodies of CAR and integrin. Results: The highest CAR expression was found in SMMC-7721 and A549 cell lines, the lowest in K562 cells. The results of fluorescence microscope and flow cytometry suggested that Ad5 had the highest efficiency in infecting SMMC-7721 and A549 cell lines, and the lowest in infecting K562 cells. Conclusion：The infection efficiency of adenovirus is determined by the levels of CAR and integrin on cancer cells.
2006, 13(3):214-218.
DOI: 10.3872/j.issn.1007-385X.2006.3.013
Abstract:
Objective: To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant , laying a foundation for gene therapy research of malignant tumors. Methods: The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus; the products were cotransfered into HEK-293 cell line with helper plasmid PJM17. The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results:The -p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells (1×1011pfu/ml). The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR. Ad. NT4p53Ant had strong killing effect on HepG2 cells. Compared with Ad.GFP, Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells. Conclusion: The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques，laying a foundation for further research of gene therapy of cancer.
Objective: To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant , laying a foundation for gene therapy research of malignant tumors. Methods: The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus; the products were cotransfered into HEK-293 cell line with helper plasmid PJM17. The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results:The -p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells (1×1011pfu/ml). The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR. Ad. NT4p53Ant had strong killing effect on HepG2 cells. Compared with Ad.GFP, Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells. Conclusion: The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques，laying a foundation for further research of gene therapy of cancer.
2006, 13(3):219-220.
DOI: 10.3872/j.issn.1007-385X.2006.3.014
Abstract:
目的: 分析中国山西襄垣地区人乳头瘤病毒16型 HPV16Z 基因的结构特点。方法: 对 HPV16Z 进行双向测序,并将其基因全序列与德国标准株进行对比分析。结果: DNA序列分析表明， HPV16Z 与已发表的德国标准株基因长度相等，其中LCR和L1、E6部位的核酸片段存在变异。LCR的变异部位分别位于7431～7432 nt、7433 nt、7495 nt、7852 nt,变异方式分别为碱基插入、碱基替代及碱基缺失突变；L1的变异部位分别位于6169～6171 nt、6901～6902 nt、6949～6951 nt,其变异方式为碱基替代、碱基插入及碱基缺失突变；E6的变异部位位于350 nt，其变异方式为碱基替代突变。结论: 中国山西襄垣地区妇女宫颈癌患者组织中HPV16型病毒 HPV16Z 基因结构与德国标准株 HPV16 基因之间存在一定的差异。
目的: 分析中国山西襄垣地区人乳头瘤病毒16型 HPV16Z 基因的结构特点。方法: 对 HPV16Z 进行双向测序,并将其基因全序列与德国标准株进行对比分析。结果: DNA序列分析表明， HPV16Z 与已发表的德国标准株基因长度相等，其中LCR和L1、E6部位的核酸片段存在变异。LCR的变异部位分别位于7431～7432 nt、7433 nt、7495 nt、7852 nt,变异方式分别为碱基插入、碱基替代及碱基缺失突变；L1的变异部位分别位于6169～6171 nt、6901～6902 nt、6949～6951 nt,其变异方式为碱基替代、碱基插入及碱基缺失突变；E6的变异部位位于350 nt，其变异方式为碱基替代突变。结论: 中国山西襄垣地区妇女宫颈癌患者组织中HPV16型病毒 HPV16Z 基因结构与德国标准株 HPV16 基因之间存在一定的差异。
2006, 13(3):221-223.
DOI: 10.3872/j.issn.1007-385X.2006.3.015
Abstract:
目的： 探索半抗原\[二硝基氟苯(DNP)\]修饰的恶性黑素瘤细胞(恶黑)激活树突状细胞(DC)体外诱导特异性T细胞反应的抗肿瘤效应。方法: 采用DNP修饰恶黑细胞B16(H-2 b)，然后在体外激活C57BL/6小鼠(H-2 b)外周血来源的DC,观察其诱导T细胞的增殖和特异性T细胞杀伤功能;在小鼠皮肤迟发性超敏反应实验和荷瘤实验中观察DNP修饰瘤苗+DC的特异性。结果: 经DNP修饰的B16细胞激活的DC,诱发T细胞增殖能力和对B16细胞的特异性杀伤效应均明显高于未修饰的B16细胞和DC。动物实验显示DNP修饰瘤苗联合DC具有明显的诱发皮肤迟发性超敏反应作用和抑瘤作用。结论: DNP修饰B16所激活的DC可以诱导更强的恶黑特异性T细胞效应。
目的： 探索半抗原\[二硝基氟苯(DNP)\]修饰的恶性黑素瘤细胞(恶黑)激活树突状细胞(DC)体外诱导特异性T细胞反应的抗肿瘤效应。方法: 采用DNP修饰恶黑细胞B16(H-2 b)，然后在体外激活C57BL/6小鼠(H-2 b)外周血来源的DC,观察其诱导T细胞的增殖和特异性T细胞杀伤功能;在小鼠皮肤迟发性超敏反应实验和荷瘤实验中观察DNP修饰瘤苗+DC的特异性。结果: 经DNP修饰的B16细胞激活的DC,诱发T细胞增殖能力和对B16细胞的特异性杀伤效应均明显高于未修饰的B16细胞和DC。动物实验显示DNP修饰瘤苗联合DC具有明显的诱发皮肤迟发性超敏反应作用和抑瘤作用。结论: DNP修饰B16所激活的DC可以诱导更强的恶黑特异性T细胞效应。
2006, 13(3):224-226.
DOI: 10.3872/j.issn.1007-385X.2006.3.016
Abstract:
Fas，又名ＣＤ９５或ＡＰＯ-１，是细胞凋亡信号受体，通过与ＦasL结合诱导细胞凋亡。肿瘤细胞既可通过表达FasL的Ｔ细胞凋亡而逃逸免疫监视；又可通过cFLIP调控ＤＩＳＣ水平，上调凋亡抑制基因及诱导Ｆas基因突变等抑制Ｆas介导的细胞凋亡。基于上述机制，可从免疫细胞层面和肿瘤细胞层面两方面调节肿瘤免疫治疗。免疫细胞抗肿瘤凋亡作用可采用激动性抗体阻断其Fas表达、 FasL 基因或 Fas 反义寡核苷酸或抗凋亡基因的转入、利用细胞因子保护作用及Caspase蛋白酶抑制剂阻断其凋亡。在肿瘤细胞方面，应用抗 Fas 抗体、 FasL 反义寡核苷酸、 Fas 或 Ｂax 基因转入肿瘤细胞及某些化疗药物与细胞因子等作用，增加肿瘤细胞Fas敏感性，使之容易被免疫细胞诱导凋亡。
Fas，又名ＣＤ９５或ＡＰＯ-１，是细胞凋亡信号受体，通过与ＦasL结合诱导细胞凋亡。肿瘤细胞既可通过表达FasL的Ｔ细胞凋亡而逃逸免疫监视；又可通过cFLIP调控ＤＩＳＣ水平，上调凋亡抑制基因及诱导Ｆas基因突变等抑制Ｆas介导的细胞凋亡。基于上述机制，可从免疫细胞层面和肿瘤细胞层面两方面调节肿瘤免疫治疗。免疫细胞抗肿瘤凋亡作用可采用激动性抗体阻断其Fas表达、 FasL 基因或 Fas 反义寡核苷酸或抗凋亡基因的转入、利用细胞因子保护作用及Caspase蛋白酶抑制剂阻断其凋亡。在肿瘤细胞方面，应用抗 Fas 抗体、 FasL 反义寡核苷酸、 Fas 或 Ｂax 基因转入肿瘤细胞及某些化疗药物与细胞因子等作用，增加肿瘤细胞Fas敏感性，使之容易被免疫细胞诱导凋亡。
2006, 13(3):227-229.
DOI: 10.3872/j.issn.1007-385X.2006.3.017
Abstract:
肿瘤可诱导机体免疫系统对其产生免疫耐受，而免疫调节酶吲哚胺2，3双加氧酶（Indoleamine 2，3-dioxygenase ，IDO）可能参与了这种免疫耐受。IDO是色氨酸代谢的限速酶,IFN能影响IDO的表达。IDO可能通过初始免疫应答和效应阶段两个环节来调节肿瘤免疫耐受。在初始免疫应答反应阶段，可能活化的调节性T细胞表达的CTLAA诱导了IDO的表达，肿瘤可能通过IDO来破坏机体的免疫系统从而对其产生免疫耐受。在效应阶段，肿瘤细胞自身表达的IDO可对肿瘤产生局部免疫耐受，其机制可能主要是通过降解局部组织微环境中的色氨酸来抑制效应T细胞的增殖,这两个环节对肿瘤免疫耐受的调节是相辅相成的。IDO抑制剂1-MT可使T细胞增殖抑制得到恢复,有望成为一种有效的抗肿瘤药物。
肿瘤可诱导机体免疫系统对其产生免疫耐受，而免疫调节酶吲哚胺2，3双加氧酶（Indoleamine 2，3-dioxygenase ，IDO）可能参与了这种免疫耐受。IDO是色氨酸代谢的限速酶,IFN能影响IDO的表达。IDO可能通过初始免疫应答和效应阶段两个环节来调节肿瘤免疫耐受。在初始免疫应答反应阶段，可能活化的调节性T细胞表达的CTLAA诱导了IDO的表达，肿瘤可能通过IDO来破坏机体的免疫系统从而对其产生免疫耐受。在效应阶段，肿瘤细胞自身表达的IDO可对肿瘤产生局部免疫耐受，其机制可能主要是通过降解局部组织微环境中的色氨酸来抑制效应T细胞的增殖,这两个环节对肿瘤免疫耐受的调节是相辅相成的。IDO抑制剂1-MT可使T细胞增殖抑制得到恢复,有望成为一种有效的抗肿瘤药物。
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.