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ABSTRACT: To investigate the effect of miR-146a on the Th1/Th2 cytokine expression in mouse RAW264.7 cell line and primary peritoneal macrophage.
miR-146a mimics, mimics negative control (NC mimics), inhibitor miR-146a and inhibitor negative control (NC inhibitor) were transfected into RAW264.7 cells and freshly isolated peritoneal macrophage. IL-18, IL-5 and IL-10 expressions in the cells were measured by real time PCR.
MiR-146a mimics suppressed IL-18 expression (P<0.05), and miR-146a specific inhibitor increased IL-18 expression significantly (P<0.05). However, IL-5 and IL-10 expressions were not affected by both miR-146a mimics and miR-146a inhibitor transfections.
These data demonstrate at first time that miR-146a can regulate Th1 cytokine IL-18 expression, but not affect Th2 cytokine IL-5 and IL-10 expressions.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2011; 27(5):477-9.
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ABSTRACT: To establish an human-mouse chimeric antibody against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (death receptor 5, DR5) in an eukaryotic cell line and analyse its tumoricidal activity.
The cDNAs encoding for the variable regions of heavy chain (V(H);) and light chain (V(L);) of AD5-10 were amplified by PCR and inserted into the human IgG heavy and light chain containing expression vector RpCI-neo, respectively. The recombinant plasmids were co-transfected into HEK293 and/or CHO cells. The production of anti-DR5 human-mouse chimeric antibody (hmAD5-10) and the antibody affinity for DR5 were identified by ELISA and Western blot assay. The tumoricidal activity of hmAD5-10 was demonstrated by MTS assay. The stable expression cells were selected and cultured in serum-free medium.
Two stable CHO cells CHO-A5 and CHO-B11 with the chimeric antibody hmAD5-10 expression were established, in which the production of hmAD5-10 were reached at (0.36±0.11) mg/L and (0.16±0.01) mg/L, respectively. The hmAD5-10 secreted from the cells can well bind with DR5 and kill the cultured leukemia SVT35 cells by apoptosis remarkably.
The human-mouse chimeric antibody hmAD5-10 was successfully expressed in the eukaryotic cells and resulted tumor cell death by apoptosis. This study lays a fundamental basis for the potential application of the recombinant chimeric antibody in cancer therapy.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2011; 27(4):415-8.
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ABSTRACT: To express a full-length human-mouse chimeric anti-DR5 antibody from a single open reading frame with tumoricidal activity to various cancer cells.
The heavy and light chains of chimeric antibody were joined by the Furin and 2A (F/2A) self-cleavage peptide and cloned into a lentiviral vector of pWPXL. Then the HEK293 cells were infected with the constructed expression vector pWPXL-HF2AL. Western blot, enzyme-linked immunosorbent assay (ELISA) and MTS assay were used to detect the chimeric antibody expression, cleavage, binding affinity to the antigen and tumoricidal activity to various tumor cells.
The recombinant chimeric antibody was successfully expressed from a single open reading frame in pWPXL-HF2AL construct. And it possessed a similar binding affinity to the parental murine counterpoint and strong tumoricidal activity to various cancer cells. For example, on the concentration of 3 µg/ml, it made the relative cells viability of HCT116, SMMC7721, A549 and U251 down to 20.6% ± 2.6%, 35.1% ± 2.7%, 76.1% ± 6.1% and 15.6% ± 2.0% respectively.
The human-mouse chimeric anti-DR5 antibody of F/2A peptide is successfully expressed. Possessing a strong tumoricidal activity in various cancer cells, it may provide a novel strategy for cancer biotherapy.
Zhonghua yi xue za zhi 03/2011; 91(10):707-10.
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ABSTRACT: To study the controllable expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in mesenchymal stem cells and evaluate its potential tumoricidal effects in cancer therapy.
The controllable TRAIL expression vector of Ad-Tet-TRE-TRAIL was established in an adenovirus vector for transfection into murine mesenchymal stem cells. The controllable expression and secretion of TRAIL were detected by Western blot and enzyme-linked immunosorbent assay. The viability of hepatocellular carcinoma cells was determined by MTT assay. The tumoricidal activity of TRAIL was determined by Annexin-V/PI staining and flow cytometry.
The murine expression model of TRAIL was successfully established in the presence of doxycycline. The secreted TRAIL in cell culture medium could efficaciously suppress the growth of human hepatocellular carcinoma SMMC-7402 by induced apoptosis. The cell viability of SMMC-7402 was 66.5% ± 4.8% and 42.9% ± 6.5% at post-treatment versus 97.3% ± 2.2% and 99.4% ± 4.7% in the control group at 24 h and 48 h.
The controllable TRAIL expression mediated by mesenchymal stem cells kills human hepatocellular carcinoma cells effectively. And it may provide a novel therapeutic strategy for hepatocellular carcinoma.
Zhonghua yi xue za zhi 03/2011; 91(8):544-8.
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ABSTRACT: To construct the human/mouse chimeric antibody of a functional anti-death receptor 5 (DR5) antibody. Methods The viable region of light chain (VL) and viable region of heavy chain (VH) genes of anti-DR5 antibody were amplified and cloned into the light- and heavy-chain expression vectors respectively, then the recombinant plasmids were co-transfected into dihydrofolate reductase(-) Chinese hamster ovary cell (CHO-dhfr(-)) for expression. The positive clone was screened by the two selective genes (neo and dhfr). The humanization and specificity of chimeric antibody was identified by ELISA and Western blotting, and the tumoricidal activity of the expressed chimeric antibody was detected by tetrazolium salt phenazine methosulfate assay.
The expression vectors stably expressed chimeric antibody in CHO-dhfr(-). In the cell supernatant of the F4' clone, the human IgG heavy constant region and light constant region were identified. Moreover, the secreted chimeric antibody retained the binding capacity to the antigen (DR5) and decreased the cell viability of Jurkat and HCT116 cells to 73.15% and 77.30% in vitro respectively.
The human/mouse anti-DR5 chimeric antibody has been successfully expressed in eukaryotic cells and shows tumoricidal activity, which establishes a foundation for the future research of humanized antibody medicine.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 01/2009; 30(6):690-5.
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ABSTRACT: To investigate the effects of human telomerase reverse transcriptase (hTERT) promoter and survivin promoter in tumor-specific gene therapy.
hTERT promoter and survivin promoter were obtained by PCR using Jurkat genomic DNA. Recombinant adeno-associated virus (AAV) vectors containing exogenous TRAIL gene and hTERT promoter or survivin promoter were constructed and designated as rAAV-hTERT-TRAIL (h/TRAIL) or rAAV-survivin-TRAIL (s/TRAIL). rAAV particles were obtained after packing and purification and the virus titer was calculated by real-time PCR. Human hepatocellular carcinoma (HCC) cells of the lines SMMC-7721, BEL-7402, HepG2, and Hep3B, and primary human hepatocytes (PHHs) were transfected with h/TRAIL or s/TRAIL. Flow cytometry was used to detect the expression of the reporter gene enhanced green fluorescent protein (EGFP), so as to examine the activity of the two promoters. MTT method was used to detect the activity of the cells. Fifteen BalB/C mice underwent subcutaneous injection of SMMC-7721 cells so as to establish tumor models and then were randomly divided into 3 groups to undergo intra-tumor injection of h/TRAIL, s/TRAIL, or PBS. The growth of tumor was observed for 5 weeks, and then peripheral blood samples were collected to examine the serum AST and ALT levels. TUNEL was used to detect the apoptosis if the tumor cells.
All the SMMC-7721, BEL-7402, HepG2, and Hep3B cells driven by both h/TRAIL and s/TRAIL showed EGFP expression, however, no fluorescence could be seen in the PHHs transfected with h/TRAIL and s/ TRAIL. MTT method showed that 72 hours after the transfection of h/TRAIL and s/TRAIL the survival rates of the SMMC-7721, BEL-7402, and HepG2 cells all decreased, however, the survival rate of the Hep3B cells and PHHs did not changed significantly. The size of the subcutaneous tumor of the mice of the h/ TRAIL group was 625 mm3, significantly smaller than that of the PBS group (1500 mm3, P <0.05), however, the tumor size of the s/TRAIL group was 1117 mm3, not significantly different from that of the PBS group (P >0.05). The AST and ALT levels of all mice did not change significantly 5 weeks after the intratumor injection.
Tumor-specific promoters are promising candidates in targeted tumor gene therapy.
Zhonghua yi xue za zhi 02/2008; 88(7):475-9.
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ABSTRACT: To identify the binding proteins to PDZ domain of ERBIN.
Using PDZ domain of ERBIN as the bait, yeast two-hybrid technology was employed to screen the human lymphocyte leukemia cells MATCHMAKER cDNA library. The protein interaction was identified by immunoprecipitation.
A PDZ-binding protein, TAX1, was identified.
TAX1 is a novel binding protein to PDZ domain of ERBIN.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 07/2007; 29(3):307-11.
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ABSTRACT: To screen new drug for the treatment of acute promyelocytic leukemia, psoriasis and acne, high-throughput drug screening cell models marked by green fluorescent protein (GFP) have been established.
Eight repeats of retinoic acid response element (RARE) were synthesized and cloned into a GFP expression vector. This construct was stably transfected into cells in vitro. Stable and sensitive cell clones with high copy numbers of RARE were selected by retinoic acid (RA) using fluorescence-activated cell sorting (FACS).
A cell line has been chosen to be high-throughput drug screening cell model. This model was shown with low background, high sensitive and good reproducibility, and was convenient and inexpensive.
This drug screening cell model can be used for retinoic acid receptor target high-throughput drug screening.
Yao xue xue bao = Acta pharmaceutica Sinica 11/2005; 40(10):908-11.
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ABSTRACT: To study the effect of 8-chloro-adenosine (8-Cl-Ado) on the sensitivity of human hepatoma and breast cancer cell lines to TRAIL-induced apoptosis in vitro and its mechanisms.
Recombinant soluble TRAIL (rsTRAIL) or 8-Cl-Ado was used to treat hepatoma cell line BEL-7402 and breast cancer cell line MCF-7 in vitro. MTT assay was used to evaluate cell viability. The effect of cotreatment with rsTRAIL and 8-Cl-Ado was analyzed. NF-kappaB activity reporter plasmid was designed to measure the activity of transcription factor NF-kappaB. After transient transfection with the reporter plasmid, which contains NF-kappaB-responsive elements, into the cell lines, cells were treated with rsTRAIL and/or 8-Cl-Ado, then the activity of the reporter gene luciferase was determined. Different kinds of caspase inhibitors were used to measure the effect of caspases in the rsTRAIL and/or 8-Cl-Ado induced apoptosis.
8-Cl-Ado could greatly enhance sensitivity of BEL-7402 and MCF-7 cells to reTRAIL. Treatment with 8-Cl-Ado and rsTRAIL inactivated transcription factor NF-kappaB and induced apoptosis in BEL-7402, but not in MCF-7. Caspase family inhibitor could not prevent apoptosis induced by 8-Cl-Ado and rsTRAIL in BEL-7402 cells, however, it could block apoptosis in MCF-7 cells, indicating that two different apoptosis pathways in MCF-7 and BEL-7402 might exist, one was caspase dependent and the other caspase independent. Moreover, all of the inhibitors of caspse-3, -8 and -9 could not block apoptosis induced by the co-treatment.
8-chloro-adenosine can enhance the sensitivity of human hepatoma cell line BEL-7402 and breast cancer cell line MCF-7 to rsTRAIL, even though MCF-7 is TRAIL-resistant. 8-Cl-Ado combined with rsTRAIL can trigger different signal pathways in MCF-7 and BEL-7402, which are caspase dependent and independent, respectively.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 11/2005; 27(10):586-90.
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ABSTRACT: To estimate the effect of two simple filters, two or more positive peptide filter and Unified Score filter on the true positive rate of protein and peptide.
Twenty-two LC-MS/MS datasets were from 18 known protein mixture. Two or more positive peptide filter and Unified Score filter were applied to the 22 datasets. The filters effect was evaluated according to the true positive rate of protein and peptide for each filter.
The positive rates of protein and peptide from two or more peptide filter raised from 56.49% to 92.86%-99.12% (for protein) and from 90.67% to 97.74%-99.62% (for peptide), but many positive proteins were filtered out. The positive rates of protein and peptide from Unified Score (ThermoFinnigan value 2400) were only about 35.51% and 82.99%, but after adjusted the value (3900) according to the number of false positive peptide, those positive rate raised to 63.61% (for protein) and 91.97% (for peptide).
Two or more peptides requirement could significantly decrease false positive rate, but it also may filter out many true positive proteins especially low molecular weight and less abundant proteins. Unified Score may be a better filter than Xcorr and DeltaCn combination and the value of 3900 is found to be more suitable for this particular datasets.
Chinese Medical Sciences Journal 07/2005; 20(2):99-103.
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ABSTRACT: To explore the role and mechanisms of chemotherapeutic drugs in TRAIL induced cell death.
Tumoricidal activities of the chemotherapeutic drugs and/or rsTRAIL in 13 strains of tumor cell lines were evaluated by MTS-PMS assay and flow cytometry. DR5 expression in the cells was observed by Western blot.
The apoptosis of human promyelocytic leukemia cells HL-60, liver cancer cells BEL-7402, T-acute lymphoblastic leukemia cells Jurkat, and myeloid leukemia cells K562 treated with rsTRAIL at 0.5 microg/ml were 53.20%, 52.20%, 51.54%, 52.70%, and 41.00%, respectively, while that of the embryonal spleen cells 293 was 24.00%. However, the apoptosis percentages of lung cancer cells anti 973, breast cancer cells MCF-7, Chinese hamster ovarian cancer cells COS-7, neuroglialoma cells U251, neuroblastoma cells SH-SY5Y, glioma cells BT-325, rat pheochromocytoma cells PC12, and mouse adrenal epithelial cells NIH3T3 were all less than 10% under the same conditions. The sensitivity of central neuron cells of SH-SY5Y, PC-12, U251, BT3251, and human embryonal spleen cells 293, which were not sensitive to rsTRAIL challenges, increased remarkably after treatment with CHX, CP, and 8-CA at sub-toxic doses plus rsTRAIL at 0.5 microg/ml. The expressions of DR5 were up-regulated and kept pace with the onset of apoptosis in the BEL-7402 liver cancer cells.
The chemotherapeutic drugs including CHX, CP, and 8-CA at sub-toxic doses can enhance antitumor activity of rsTRAIL.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 11/2004; 26(5):524-8.
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ABSTRACT: To investigate the expression of the soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated by adeno-associated virus (AAV) and its tumoricidal activity in vitro and vivo.
The recombinant AAV expression vector encoding the extracellular domain (114-281aa peptide, TRAIL(114-281)) of TRAIL was constructed and transfected into human embryotic kidney cells HEK293 for virus package. The human tumor cell lines of T lymphocyte leukemia Jurkat, liver cancer HepG2 and SMMC-7721, and cervical cancer HeLa were transduced by using the recombinant virus particles respectively. The recombinant virus particles were also injected into C57BL/6 mice via the hepatic portal vein or hypodermic, intramuscular, celiac and oral pathways to study the expression of TRAIL(114-281). The recombinant virus titer was determined by real-time PCR. The expression of TRAIL(114-281) was evaluated by ELISA, Western blotting and immunohistochemistry assay respectively. The tumoricidal activity and apoptosis were evaluated by MTT assay.
The recombinant AAV encoding for the soluble TRAIL (114 - 281aa) were constructed successfully. The titer of recombinant virus was 7.5 x 10(12) genome particles (Gps)/ml. Transduction of rAAV-TRAIL(114-281) led to high level expression of TRAIL(114-281) and the induction of apoptosis of Jurkat, Hela and SMMC-7721 cancer cells, but not HepG2 cells, in vitro. The recombinant peptide TRAIL(114-281) in trimeric active form was highly and constantly expressed in the hepatocytes and secreted into the serum up to 6 months in the of C57BL/6 mice injected with the recombinant virus particles via the hepatic portal vein. The peptide TRAIL(114-281) in the livers, but not other tissues, were also detected in the mice administrated with rAAV-TRAIL(114-281) particles via subcutaneous, intramuscular, intraceliac or oral pathway.
The long term, stable and liver-tropism expression of peptide TRAIL(114-281) in mice mediated by rAAV-TRAIL(114-281) provides a prospective novel strategy for tumor gene therapy of numerous cancers, especially liver cancer.
Zhonghua yi xue za zhi 11/2004; 84(19):1635-41.
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ABSTRACT: To investigate the relationship between apoptosis induced by CD3epsilon and 5-fluorouracil (5-FU), and study the P53 expression in the apoptosis process provide a novel insight and useful information of the apoptosis signaling pathway induced by CD3epsilon and/or 5-FU, and an important implication for the treatment of T-lymphocyte leukemia.
The viabilities of Jurkat T lymphocytes (JK), TJK [JK with over-expression of the CD8epsilon chimeria molecule] and T3JK [JK with over-expression of the CD8epsilon (Y170F/Y181F) mutation molecule] cells were cultured and treated with pre-coated anti-CD8 mAb (200 micro g/ml) and/or 5-FU (2.5 micro g/ml) were detected with MTS assay and the apoptosis percentages were calculated. Western blot was used to detect P53 expression. To confirm the role of P53 in 5-FU-treated T lymphocytes, pCMV-p53 plasmid with wild type or mutant p53 were co-transfected transiently with pEGFP-c1 into TJK and T3JK cells, respectively.
CD3epsilon or 5-FU induced apoptosis of TJK with increase of P53 expression. Co-treatment with CD3epsilon specific antibody and 5-FU elevated the apoptotic rates and P53 expression in TJK cells remarkably. The cells transfected with wild-type p53 exhibited more sensitivity to 5-FU than that transfected with mutant p53.
Co-treatment of CD3epsilon and 5-FU increases the apoptosis and p53 expression, suggesting that there is a synergetic role of CD3epsilon and 5-FU on T lymphocytes.
Zhonghua yi xue za zhi 12/2003; 83(22):1962-7.
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ABSTRACT: To clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4).
The yeast two-hybrid system was used for this study. Automatic sequencing was carried out for DNA sequencing. The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively. Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells.
Two positive clones, named as pADB1 and pADB2, were obtained. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97%. The insert of pADB2 shared no homology with any known peptides in GeneBank. Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically.
FPRL1 may associate with DR4CD in vivo specifically. The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 07/2002; 24(3):310-4.
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ABSTRACT: To identify the genes differentially expressed in leukemia cell apoptosis induced by recombinant soluble tumor necrosis factor-related apoptosis inducing ligand (rsTRAIL).
Suppression subtractive hybridization (SSH) and polymerase chain reaction (PCR) were used for the cloning and identification of the genes differentially expressed in the apoptotic Jurkat cells induced by TRAIL. Slot blot and Northern blot were used for the expression pattern analysis of the genes. Automatic DNA sequencing was used for DNA sequence analysis.
Six cDNA fragments differentially expressed in the Jurkat leukemia cells treated with TRAIL were found, in which four were inhibited and two were activated during the Jurkat cell apoptosis treated with TRAIL. Among which the five genes of A14, X1, D1, A23 and C5 were found at the first time by DNA sequencing and GeneBank database searching. So that they were registered in GeneBank as AW731601, AW731602, AW731603, AW731604 and BE239235, respectively. It was found that the gene D1 was expressed higher in Jurkat leukemia cells and MCF-7 breast cancer cells than that in K562 leukemia, 825 gastric cancer and 7721 liver cancer cells.
Five novel cDNA fragments were found, and among which D1 might be a tumor specific gene.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 07/2002; 24(3):238-41.
