ABSTRACT
<p><b>OBJECTIVE</b>To investigate whether the major histocompatibility complex class I chain-related gene A gene (MICA) polymorphism and serum soluble MICA level were associated with the occurrence and development of colorectal cancer.</p><p><b>METHODS</b>DNA samples from 117 colorectal cancer patients and 113 healthy individuals from Yangzhou in Jiangsu province were genotyped by using the polymerase chain reaction (PCR) and sequence-specific primer (SSP) method and PCR based sequencing. In addition, polymorphism at position 129 was also analyzed by PCR-SSP. Serum levels of soluble MICA were measured by a sandwich ELISA method.</p><p><b>RESULTS</b>Neither the extracellular nor the transmembrane region polymorphisms of MICA gene were associated with the occurrence and the different stages of colorectal cancer. In contrast, the frequency of the methionine residue at position 129 was significantly decreased in the patient group. Soluble MICA levels in sera were increased in the late stages of colorectal cancer.</p><p><b>CONCLUSION</b>Although there was no genetic susceptibility attributed to MICA gene polymorphism with regard to development of colorectal cancer, serum levels of soluble MICA may be a diagnostic marker of advanced stages.</p>
Subject(s)
Female , Humans , Male , Colorectal Neoplasms , Blood , Genetics , Enzyme-Linked Immunosorbent Assay , Genotype , Histocompatibility Antigens Class I , Blood , Genetics , Polymerase Chain Reaction , Polymorphism, Genetic , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the influence of mIL-7 on the immune response induced by vaccine of bcr-abl fusion gene fragment in mouse.</p><p><b>METHODS</b>BALB/c mice were immunized by i. m. injection of pVbcr-abl/mIL-7 and pVbcr-abl, respectively. The specific antibody to p210bcr-abl protein was assayed by ELISA. The CTL activity of spleen cells from the immunized mice was assessed with LDH release test.</p><p><b>RESULTS</b>The pVbcr-abl/mIL-7 and pVbcr-abl-immunized BALB/c mice elicited higher specific antibodies to p210bcr-abl protein. The specific antibody level of former group was higher than that in latter group, but the difference was statistically not significant. The spleen cells from the immunized mice showed more effective CTL activity than that from control group. The cytotoxic activity of spleen CTLs induced by pVbcr-abl/mIL-7 immunized mice exceeded that of pVbcr-ab-immunized mice.</p><p><b>CONCLUSION</b>The mIL-7 may influence the growth and differentiation of T cells, promote some T cells migrating into tumor tissue and up-regulate the specific cellular immune response. The results of this study provided an useful experimental basis for preclinical research on gene vaccine for chronic myeloid leukemia.</p>
Subject(s)
Animals , Female , Humans , Mice , Antibodies , Blood , Cancer Vaccines , Genetics , Allergy and Immunology , Cell Line, Tumor , Cytotoxicity, Immunologic , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Fusion Proteins, bcr-abl , Genetics , Allergy and Immunology , Interleukin-7 , Genetics , Allergy and Immunology , K562 Cells , Mice, Inbred BALB C , Random Allocation , Spleen , Cell Biology , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Vaccination , Vaccines, DNA , Allergy and ImmunologyABSTRACT
To study the influence of vaccine of bcr-abl fusion gene fragment on inoculated SP2/0/bcr-abl tumor cells in mice, BALB/c mice were immunized with pVbcr-abl, pVbcr-abl/mIL7 plasmids, respectively, then SP2/0/bcr-abl cells expressing the fragment of bcr-abl fusion gene were inoculated subcutaneously into the groin of BALB/c mice in order to observe the effect of vaccine on growth of inoculated SP2/0/bcr-abl tumor cells. The results showed that there were distinct differences on the time of tumor growth, the time of tumor ulceration, tumor volume and survival time of mice bearing tumor between two immunized groups and two control groups (blank and vacant plasmid groups). The mice immunized with pVbcr-abl/mIL7 lived longer as compared to mice immunized with pVbcr-abl. The tissue of inoculated tumor was more compact, tumor organ was larger, tumor form was irregular in 2 control groups, while the tissue of inoculated tumor was looser, tumor volume was smaller, and with mass inflammatory infiltration in two immunized groups. Moreover, the metastatic tumor cells were found in the livers of control groups, but not observed in two immunized groups. It is concluded that the protection occurred in immunized mice which inhibited the growth of SP2/0/bcr-abl tumor cell in vivo.
Subject(s)
Animals , Female , Mice , Cancer Vaccines , Allergy and Immunology , Metabolism , Cell Line, Tumor , Fusion Proteins, bcr-abl , Genetics , Allergy and Immunology , Mice, Inbred BALB C , Multiple Myeloma , Genetics , Allergy and Immunology , Pathology , Neoplasm Transplantation , Random Allocation , Vaccines, DNA , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To study the specific immune response induced by a recombinant eukaryotic expression plasmid encoding bcr-abl fusion gene fragment so as to explore new immunotherapy in mouse.</p><p><b>METHODS</b>A recombinant eukaryotic vector pVbcr-abl expression cDNA fragment of bcr-abl fusion gene was constructed and used to immunize BALB/c mice. Serum level of bcr-abl specific antibody was detected by enzyme-linked immunosorbent assay (ELISA). Twenty days later the immunized mice were subcutaneously inoculated SP2/0/bcr-abl cells. The survival time, tumor growth time and lymphocytic infiltration were observed. T cells infiltration into tumor tissue was analyzed by immunohistochemistry. Changes of T cell subset in the spleen of mice was analyzed by fluorescent-activated cell sorting (FACS) and the cytotoxicity T lymphocyte (CTL) activity in spleen by lactate dehydrogenase (LDH)-release assay.</p><p><b>RESULTS</b>The eukaryotic expression vector pVbcr-abl was constructed successfully, and highly expressed the cDNA fragment of bcr-abl fusion gene. The BALB/c mice immunized with the vector could generate the specific antibody and CTL, resulting in a specific immunoprotection. There were dramatic differences in the tumor-forming time, tumor ulcer appearing time and tumor-growing speed between the immunized and the control groups. The mice had longer survival time in the immunized group than in the control group. There were a large amount of CD3(+) T cells infiltration in tumor tissue of the immunized mice. The spleen cells from the immunized mice had higher CTL activity with a alteration of T cell subset, the CD4(+)/CD8(+) ratio being 1.54 +/- 0.29, higher than that of control group (1.18 +/- 0.30).</p><p><b>CONCLUSION</b>The recombinant eukaryotic expression plasmid pVbcr-abl can induce in vivo not only the generation of specific antibody, but also high level of specific CTL activity, resulting in killing the SP2/0/bcr-abl tumor cells directly and inhibiting the tumor growth.</p>
Subject(s)
Animals , Female , Mice , Fusion Proteins, bcr-abl , Genetics , Gene Expression , Genetic Vectors , Immunotherapy , Mice, Inbred BALB C , Plasmids , Genetics , Random Allocation , TransfectionABSTRACT
To establish SP2/0 cell line H-2(d) stably expressing bcr-abl fusion gene fragment, the bcr-abl fusion gene was subcloned into retroviral vector pLXSN from pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with the help of lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected to determine viral titer, the viral titer was 2 x 10(7) CFU/ml. The SP2/0 cells were infected with the collected viral supernatant. The results showed that after G418 selection, the bcr-abl fusion gene was integrated into the chromosome of SP2/0 cells infected stably, with recombinant retrovirus and expressed in SP2/0 cells confirmed by PCR and RT-PCR respectively. In conclusion, the mouse tumor cell lines expressing the bcr-abl fusion protein were successfully established and would be used as a experimental cell model for anti-CML immunotherapy.
Subject(s)
Animals , Humans , Mice , Cell Line , Cell Line, Tumor , Fusion Proteins, bcr-abl , Genetics , Gene Expression Regulation, Neoplastic , Genetic Vectors , Genetics , K562 Cells , Mice, Inbred BALB C , Multiple Myeloma , Genetics , Pathology , NIH 3T3 Cells , Peptide Fragments , Genetics , RNA, Messenger , Genetics , Retroviridae , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , MethodsABSTRACT
To clone the differential genes in antibiotic resistence Neisseria gonorrhoeae,the library that contains the fragments of differential genes between antibiotic resistance strain and standard reference strain of Neisseria gonorrhoeae was constructed which used suppression subtractive hybridization technique. Then the antibiotic resistance relational genes fragments were cloned and analyzed. Antibiotic resistance Neisseria gonorrhoeae subtractive library that has high subtractive efficiency was set up successfully. The amplified library contained 2500 positive clones. Sequence analysis was performed to find the fragments of antibiotic resistance relational genes. Five sequences were unknown previously. The fragments of antibiotic resistance genes may provide an important clue for studying the mechanism of occurrence and development of antibiotic resistance Neisseria gonorrhoeae.