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1.
Chinese Medical Journal ; (24): 4434-4439, 2012.
Article in English | WPRIM | ID: wpr-331358

ABSTRACT

<p><b>BACKGROUND</b>Papillary thyroid carcinoma (PTC) represents one of the most frequent endocrine malignancies. Several factors have been found to be involved in determining the outcome of treatment for patients with PTC. Large tumor size, diagnosis at an early age, extra-thyroidal invasion, aggressive histological variants, and distant metastases are the most important determinants of a poor outcome. BRAF(V600E) mutation has been found to be a major genetic alteration in PTC. This study aimed to evaluate progression in patients with multifocal and solitary PTC.</p><p><b>METHODS</b>We performed a retrospective study to analyze 368 patients with PTC who underwent surgery, including 282 patients with solitary PTC and 86 patients with multifocal PTC. The status of BRAF(V600E) mutation in all tumor foci from multifocal PTC was detected.</p><p><b>RESULTS</b>Our study suggested that multifocal PTC was more related to lymph node metastasis and vascular invasion than solitary PTC. However, the distant metastasis rate and 10-year survival rate showed no difference between these two groups. The number of tumor foci did not affect progression of disease in multifocal PTC patients. Lymph node metastasis in multifocal PTC patients was associated with larger tumors, diagnosis at early stage, and extra-thyroidal invasion.</p><p><b>CONCLUSION</b>The status of BRAF(V600E) mutation was more frequent in multifocal PTC patients with lymph node metastasis and diagnosis at later age.</p>


Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma , Genetics , Pathology , Carcinoma, Papillary , Genetics , Pathology , Mutation , Proto-Oncogene Proteins B-raf , Genetics , Retrospective Studies , Thyroid Neoplasms , Genetics , Pathology
2.
Chinese Journal of Oncology ; (12): 513-516, 2011.
Article in Chinese | WPRIM | ID: wpr-320183

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression of indoleamine 2, 3-dioxygenase (IDO) in breast cancer and its correlation with clinicopathologic factors and prognosis.</p><p><b>METHODS</b>The expression of IDO, CD31, CD105 proteins in 40 specimens of breast cancer were assessed by immunohistochemistry.</p><p><b>RESULTS</b>The overexpression rate of IDO in breast cancer was 67.5% (27/40), and expression of IDO was closely associated with clinical stage and lymph nodes metastasis. The disease-free survival rate in patients with IDO overexpression was not significantly lower than that in patients with negative or low expression of IDO (P > 0.05). Moreover, the expression of IDO was positively correlated with CD105-labeled microvessel density (r = 0.659, P < 0.05).</p><p><b>CONCLUSIONS</b>Expression of IDO is associated with clinical stage and lymph nodes metastasis, and microvessel densitty. IDO expression may promote the growth and metastasis of breast cancer, probably via the increased agiogenesis. A larger sample study is needed to verify whether the prognosis of beast cancer is significantly correlated with IDO expression.</p>


Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Adenocarcinoma , Allergy and Immunology , Pathology , Antigens, CD , Metabolism , Breast Neoplasms , Allergy and Immunology , Pathology , Carcinoma, Ductal, Breast , Allergy and Immunology , Pathology , Carcinoma, Medullary , Allergy and Immunology , Pathology , Disease-Free Survival , Endoglin , Follow-Up Studies , Immunohistochemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase , Metabolism , Lymphatic Metastasis , Microvessels , Allergy and Immunology , Neoplasm Staging , Platelet Endothelial Cell Adhesion Molecule-1 , Metabolism , Receptors, Cell Surface , Metabolism , Survival Rate
3.
Chinese Medical Journal ; (24): 2215-2220, 2010.
Article in English | WPRIM | ID: wpr-237477

ABSTRACT

<p><b>BACKGROUND</b>S100A8 and S100A9 are two members of the S100 protein family characterized by the presence of two Ca2+-binding sites of the EF-hand type. Previous studies suggested that the whole S100 family displays significant functions in tumor growth, progression and invasion. This study aimed to determine the expression of the two indices of the family, S100A8 and S100A9, in lung cancer tissues and normal lung tissues and its correlation with clinical features.</p><p><b>METHODS</b>A total of 60 cases with a variety of clinical data that were diagnosed with different histological subtypes of lung cancer were investigated. Semi-quantitative reverse transcriptase-PCR (Sq-Rt-PCR) and immunohistochemical staining of cancer, adjacent and peripheral lung tissues were executed to distinguish the expression patterns of S100A8 and S100A9 and to further clarify their correlation with clinical features.</p><p><b>RESULTS</b>Immunohistochemical staining of both proteins showed a significant up-regulation in lung cancer tissue (S100A8, S100A9, P<0.0001), and PCR revealed that the levels of S100A8 and S100A9 expression were significantly higher in lung cancer tissues (S100A8 P=0.002/0.004; S100A9 P=0.022/0.026). The higher expression was found to be correlated with the clinical characteristics of adenocarcinoma, inflammation and stage IV lesion.</p><p><b>CONCLUSIONS</b>S100A8, S100A9 up-regulation was found in the lung adenocarcinoma and end stage lung cancer tissue, the correlation of which with their higher expression in inflammatory lung tissues may indicate the collaborative effect of inflammation on the progression of cancer.</p>


Subject(s)
Aged , Female , Humans , Male , Middle Aged , Adenocarcinoma , Genetics , Metabolism , Pathology , Calgranulin A , Genetics , Metabolism , Calgranulin B , Genetics , Metabolism , Immunohistochemistry , Inflammation , Genetics , Metabolism , Pathology , Lung Neoplasms , Genetics , Metabolism , Pathology , Reverse Transcriptase Polymerase Chain Reaction
4.
Chinese Journal of Oncology ; (12): 659-662, 2010.
Article in Chinese | WPRIM | ID: wpr-293532

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the influence of mutual interactions between FasL expressed by colon carcinoma cells and endogenous cytokines interleukin-18 on liver metastasis and invasion of human colon cancer cells.</p><p><b>METHODS</b>Using immunohistochemical streptavidin-biotin complex (SABC) method, the expressions of Fas receptor and Fas ligand in SW620 colon carcinoma cells and Chang liver cells were observed so as to provide morphological evidence for the functions of Fas receptor and Fas ligand. In an effort to examine the cytotoxicity of effector cells, CytoTox(96) Non-Radioactive Cytotoxicity Assay was adopted to measure the LDH releasing value after the SW620 cells were co-cultured with Chang liver cells.</p><p><b>RESULTS</b>It was shown that the Fas ligand of colon carcinoma SW620 cells was positive and the positive substances were distributed in the cell membrane and cytoplasm, and the Fas receptor of colon carcinoma SW620 cells was negative. The Fas receptor of Chang liver cells turned out to be positive and the positive substances were distributed in the cell membrane, and the Fas ligand of Chang liver cells was negative. At 6 hours after co-culture of IFN-γ-stimulated Chang liver cells with interleukin-18-stimulated (for 36 h) SW620 cells or unstimulated SW620 cells, the cytotoxicity of SW620 cells to IFN-stimulated Chang liver cells at effector-to-target ratios of 10:1, 5:1, 2.5:1 and 1.25:1 was 68.3%, 49.8%, 21.1%, 9.7% (F = 76.87, P < 0.05) and 32.7%, 21.8%, 11.1%, 6.7% (F = 7.27, P < 0.05), respectively. The non-radioactive cytotoxicity assay showed that the apoptotic rate of Chang liver cells was remarkably increased with the increase of planting concentration of SW620 after the SW620 cells were co-cultured with Chang liver cells. The cytotoxicity was significantly enhanced by interleukin-18.</p><p><b>CONCLUSION</b>The FasL expression of human colon cancer cells may be regulated by endogenous interleukin-18 in the host microenvironment and enhance the liver colonization competence of colon cancer cells through induction of apoptosis in the Fas-expressing hepatocytes.</p>


Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Colonic Neoplasms , Allergy and Immunology , Metabolism , Pathology , Cytotoxicity, Immunologic , Fas Ligand Protein , Metabolism , Hepatocytes , Cell Biology , Metabolism , Interferon-gamma , Pharmacology , Interleukin-18 , Pharmacology , L-Lactate Dehydrogenase , Metabolism , fas Receptor , Metabolism
5.
Journal of Experimental Hematology ; (6): 164-169, 2009.
Article in Chinese | WPRIM | ID: wpr-302174

ABSTRACT

This study was aimed to study the potential effects of alloreactive NK cells (allo-NKs) in therapy of relapsed lung cancer after haploidentical hematopoietic stem cell transplantation using donor lymphocyte infusion (DLI). The F1 donors derived-NK cells were purified with MACS magnetic separation system, in which the proportion of the alloreactive Ly49A(+) cells was detected by flowcytometry and alloreactivity was measured by LDH method. The relapse model of lung cancer after haploidentical-HSCT was established. The distribution kinetic of infused donor lymphocytes in vivo was analyzed. The inhibition of relapse tumor, infiltration of lymphocytes in situ and fluctuation of 22 kinds of cytokines in serum after DLI were compared among different groups. The results showed that the infused donor cells of allo-NK group were accumulated mostly in lung, spleen and kidney for more than 48 hours with considerable higher levels according to the distribution kinetic curve. The sizes of relapse tumors between chemotherapy + PBS group and chemotherapy + DLI group showed no difference. However, the relapsed tumors in allo-NK + DLI group were significantly smaller than that in chemotherapy + DLI group or allo-NK + PBS group, in which increased infiltration of lymphocytes were defined in situ. The levels of cytokines such as MCP-1, IL-17, IL-12 and MCP-5 in serum of allo-NK + DLI group ascended compared with control group, though the level of IL-10 declined simultaneously. It is concluded that allo-NKs prolong the survival time of infused donor lymphocytes in vivo, promote the secretion of inflammatory cytokines and Th1-type of cytokines, and further improve the antitumor effects of DLI against relapse after transplantation.


Subject(s)
Animals , Mice , Cytokines , Blood , Hematopoietic Stem Cell Transplantation , Methods , Killer Cells, Natural , Cell Biology , Lung Neoplasms , Therapeutics , Lymphocyte Transfusion , Methods , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Recurrence, Local , Therapeutics , Transplantation Conditioning , Methods
6.
Chinese Journal of Oncology ; (12): 808-812, 2007.
Article in Chinese | WPRIM | ID: wpr-298506

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the anti-tumor effects and mechanism of tumor vaccines and whether chemotherapeutic agents administered prior to immunotherapy could augment the efficacy of the vaccines.</p><p><b>METHODS</b>C57/BL mice inoculated with Lewis lung cancer cells were used as tumor models. Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene modified LA795 and Lewis lung cancer cell lines were administered as allogeneic and autologous tumor vaccines, respectively. After Lewis cells (1 x 10(7)) inoculation, the mice received irradiated GM-CSF secreting cancer vaccine solely or in combination with carboplatin. The survival of the mice was observed. The cytotoxicity of spleen cells or purified CD8(+) cells was analyzed by lactate dehydrogenase (LDH) assay. Serum level of IL-4 and IFN-gamma was detected using ELISA method.</p><p><b>RESULTS</b>The cytotoxicity of the spleen cells or purified CD8(+) T cells against Lewis cells in the mice immunized with cancer cell vaccine was significantly increased, relative to that of the control, untreated group (P < 0.05). Serum level of Th1-type cytokine IFN-gamma was increased after vaccination, whereas Th2-type cytokine IL-4 showed no significant change. The GM-CSF secreting cancer cell vaccine had no significant influence on the survival of the mice with established heavy tumor burden. The combination of chemotherapy and cancer vaccine could statistically prolong the survival time; whereas any method itself had no significant effect.</p><p><b>CONCLUSION</b>The GM-CSF secreting cancer cell vaccine can induce immune responses. The chemotherapeutic agents may be beneficial to enhance the anti-tumor activity of cancer vaccine.</p>


Subject(s)
Animals , Female , Mice , Antineoplastic Agents , Therapeutic Uses , Cancer Vaccines , Therapeutic Uses , Carboplatin , Therapeutic Uses , Carcinoma, Lewis Lung , Blood , Metabolism , Pathology , Therapeutics , Cell Line, Tumor , Granulocyte-Macrophage Colony-Stimulating Factor , Genetics , Metabolism , Interferon-gamma , Blood , Interleukin-4 , Blood , Lung Neoplasms , Metabolism , Pathology , Mice, Inbred C57BL , Transfection
7.
Journal of Experimental Hematology ; (6): 1013-1018, 2007.
Article in Chinese | WPRIM | ID: wpr-318799

ABSTRACT

This study was aimed to investigate the feasibility of low dose of fludarabine, cyclophosphamide combined with donor derived alloreactive NK cells as a new nonmyeloablative conditioning regimen in the haploidentical hematopoietic stem cell transplantation (haploidentical HSCT). F1 derived-NK cells were enriched with MACS magnetic separation system, in which the proportions of the Ly49C+ and Ly49A+ cells were detected by flow cytometry and the alloreactivity was measured by LDH method. The haploidentical HSCT models were constructed, and the myeloablativity in vivo, donor engraftment and the intensity of GVHD were compared between different myeloablative and nonmyeloablative conditioning regimens, including 9 Gy TBI, 6.5 Gy TBI, flu + cy, and flu + cy + allo-NK. The results showed that the flu + cy + allo-NK conditioning was nonmyeloablative, but the rate of donor chimerism after haploidentical HSCT was significantly higher as compared with other nonmyeloablative methods, which were (28.70 +/- 5.90)% in bone marrow and (46.40 +/- 5.00)% in spleen at day 21 post-transplantation. When compared with the flu + cy conditioning, the intensity of GVHD was slight in the flu + cy + allo-NK group, in which only a half of C57BL/6 recipients experienced weight loss, and no distinct pathological damages observed in the liver, intestine, kidney and skin samples. It is concluded donor derived-alloreactive NK cells can facilitate engraftment of the haploidentical hematopoietic stem cells and mitigate GVHD. The flu + cy + allo-NK conditioning provides a new method for those elder patients with high-risk solid tumor undergoing haploidentical-HSCT.


Subject(s)
Animals , Female , Mice , Cyclophosphamide , Graft vs Host Disease , Haplotypes , Hematopoietic Stem Cell Transplantation , Methods , Killer Cells, Natural , Transplantation , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Animal , Transplantation Chimera , Transplantation Conditioning , Methods , Vidarabine , Whole-Body Irradiation
8.
Chinese Journal of Oncology ; (12): 922-926, 2007.
Article in Chinese | WPRIM | ID: wpr-348172

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the distribution of CD4+ CD25+ regulatory T-cells (T-regs) in tumor-draining lymph nodes (TDLN) in patients with non-small cell lung caner (NSCLC), and to investigate the effect of CD4+ CD25+ T regulatory cells on the immune status of TDLN and the progression of NSCLC.</p><p><b>METHODS</b>Regional tumor-draining lymph nodes of 53 NSCLC patients were resected during the operation. The percentage of CD4+ CD25+ T-regs as a subset of CD4+ T cells and CD8+ T cells were detected by immunofluorescence and regular immunohistochemistry, respectively. The level of cytokines TGF-beta1 and IL-10 was detected by real time quantitative RT-PCR.</p><p><b>RESULTS</b>CD4+ CD25+ T-regs in tumor-infiltrating lymph nodes from the patients with NSCLC accounted for 28.80% +/- 8.06% of total CD4+ T cells, and were significantly increased comparing with that (15.48% +/- 4.66%) in the tumor-free lymph nodes (P < 0.01). The percentage of CD4+ CD25+ T-regs in TDLN of NSCLC patients was negatively correlated with the amount of CD8+ T cells within the lymph nodes (r = -0. 756, P < 0.001), but positively correlated with the level of TGF-beta1 (r = 0.645, P < 0.001) and IL-10 (r = 0.769, P < 0.001). It also increased as NSCLC getting progressed, which was 30.42% +/- 7.47% in stage III versus 16.22% +/- 4.88% in stage I and III; 32.58% +/- 7.52% in N2 versus 22.76% +/- 4.67% in N1, with a significant difference between the two groups, respectively (P < 0.01).</p><p><b>CONCLUSION</b>The population of CD4+ CD25+ T regulatory cells in tumor-draining lymph nodes in patients with non-small cell lung caner is positively correlated with the progression and infiltration of lung cancer, which might provide new immunologic method to evaluate the progression and prognosis of non-small cell lung caner. The outcomes of biotherapy for NSCLC may be improved in the future through regulating the CD4+ CD25+ T regulatory cells.</p>


Subject(s)
Aged , Female , Humans , Male , Middle Aged , CD4 Lymphocyte Count , CD8-Positive T-Lymphocytes , Pathology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Interleukin-10 , Metabolism , Lung Neoplasms , Metabolism , Pathology , Lymph Nodes , Allergy and Immunology , Metabolism , Lymphatic Metastasis , Neoplasm Staging , T-Lymphocytes, Regulatory , Pathology , Transforming Growth Factor beta1 , Metabolism
9.
Chinese Journal of Oncology ; (12): 405-410, 2007.
Article in Chinese | WPRIM | ID: wpr-255632

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene and evaluate its anti-tumor effects.</p><p><b>METHODS</b>Mouse origin TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR. The TbetaR-II extracellular domain-RANTES fusion gene was amplified by overlapping PCR method. TbetaR-II extracellular domain-RANTES fusion gene was cloned into pDC316 vector. The recombinant adenovirus vector expressing the fusion gene was constructed by adMax adenovirus vector creation system. Recombinant adenovirus vector expressing the fusion gene was transfected into LA795 cells. The expression of recombinant adenovirus was checked by Westen blot. The levels of TGF-beta1, RANTES in supernatant were checked by ELISA. The transfected cells were counted and growth curve was obtained. Apoptosis of transfected cells was detected by Annexin V FITC method. The chemotactic activity of supernatant of transfected cells to splenic lymphocytes was assayed. Transfected cells (1 x 10(5)) were inoculated into T739 mice and to observe the tumor growth and survival time. Ad-TbetaR-II extracellular domain, Ad-RANTES and Ad-TbetaR-II extracellular domain-RANTES fusion gene(1 x 10(10) pfu) were injected into the tumor in T739 mice. The tumor size and tumor weight were recorded and tumor growth inhibition rate was counted and statistically analyzed.</p><p><b>RESULTS</b>TbetaR-II extracellular domain and RANTES gene were amplified by RT-PCR and TbetaR-II extracellular domain-RANTES fusion gene amplified by overlapping PCR, were identified by DNA sequence analysis. Restriction enzyme digestion analysis showed that the recombinant vector was constructed correctly. The recombinant adenovirus vector expressing the fusion gene was constructed successfully using the AdMax Adenovirus Vector Creation System. Its titer was 8 x 10(10) pfu/ml. Ad-TbetaR-II extracellular domain-RANTES fusion gene was transfected into LA795 cells and had specific protein fragment proved by Western Blot. The concentration of TGF-beta1 was decreased and RANTES was increased in supernatant of transfected cells. The growth curve showed that recombinant adenovirus vector expressing the fusion gene could delay tumor development and induce apoptosis, with an apoptosis rate in vitro of 16.9%. The supernant of infected cells showed chemotactic activity to splenic lymphocytes. Tumor growth and survival time were prolonged significantly in group tranfected with recombinant adenovirus vector expressing the fusion gene, and tumor growth was effectively inhibited after injecting recombinant adenovirus vector expressing the fusion gene, with a tumor growth inhibition rate of 37.6%.</p><p><b>CONCLUSION</b>A recombinant adenovirus vector expressing TbetaR-II extracellular domain-RANTES fusion gene has been constructed successfully. The recombinant adenovirus vector can bind TGF-beta1 effectively, counteract immune suppression mediated by TGF-beta, enhance immune function, induce significant antitumor immune respone, inhibit tumor growth, and prolong the survival time of tumor-bearing mice.</p>


Subject(s)
Animals , Female , Humans , Mice , Adenocarcinoma , Metabolism , Pathology , Therapeutics , Adenoviridae , Genetics , Apoptosis , Cell Line , Cell Line, Tumor , Chemokine CCL5 , Genetics , Metabolism , Genetic Therapy , Methods , Genetic Vectors , Lung Neoplasms , Metabolism , Pathology , Therapeutics , Neoplasm Transplantation , Protein Serine-Threonine Kinases , Genetics , Metabolism , Random Allocation , Receptors, Transforming Growth Factor beta , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection , Transforming Growth Factor beta1 , Metabolism
10.
Chinese Journal of Cancer Biotherapy ; (6)2006.
Article in Chinese | WPRIM | ID: wpr-685642

ABSTRACT

Objective:To investigate the influence of sCD80-Linker-sCD40L fusion protein on the unspecific anti- tumor immunity in vitro.Methods:Ovarian cancer SKOV3 cells were separately transfected with recombinant adenoviral vectors containing sCD80-Linker-sCD40L fusion gene,sCD80 gene,sCD40L gene or with control adenovirus.The expres- sion of the sCD80-Linker-sCD40L fusion protein,sCD80 protein and sCD40L protein in the supernatants of SKOV3 cells was determined by ELISA.Dendritic cells(DCs)were cultured with peripheral blood mononuclear cells from a patient with ovarian carcinoma.DCs and autologous T cells were co-cuhured and were exposed to different supernatants for 48 h. The allostimulatory effects of DCs on T cells were determined by mixed lymphocyte reaction(MLR).The unspecific kill- ing activities of induced T cells against SKOV3/K562 cells were measured by LDH-releasing assay.Results:ELISA assay showed that levels of the sCD80-Linker-sCD40L fusion protein,sCD80 protein and sCD40L protein in the supernatants of transfeced SKOV3 cells were 2.791 ng/ml,1.956 ng/ml and 1.407 ng/ml,respectively.The fusion protein-exposed DCs ([0.382?0.053]vs[0.167?0.028],P

11.
Chinese Journal of Oncology ; (12): 423-425, 2005.
Article in Chinese | WPRIM | ID: wpr-358614

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the changes of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer.</p><p><b>METHODS</b>Sixty four patients with breast cancer, 15 patients with benign breast tumors and 9 healthy volunteers were included in this study. The proportion of CD(4)(+)CD(25)(+) T cells population in total T cells was evaluated by flow cytometric analysis. The cytokine production (TGF-beta1) was measured by ELISA.</p><p><b>RESULTS</b>The population of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer accounted for (5.1 +/- 2.9)% of the total amount of T lymphocytes, and was significantly higher in comparison with that in patients with benign tumors and in healthy volunteers (P < 0.05). The CD(4)(+)CD(25)(+) T cells population in breast cancer patients was positively correlated with the cancer size and with TGF-beta1 level (r = 0.511 and r = 0.253, respectively), and negatively correlated with CD(8)(+)CD(28)(+) T cells and NK cells (r = -0.243 and r = -0.301, respectively).</p><p><b>CONCLUSION</b>The CD(4)(+)CD(25)(+) regulatory T cells in peripheral blood of patients with breast cancer is significantly increased in comparison with that in patients with benign breast tumor and in healthy subjects. It may be responsible for immune suppression in breast cancer patients.</p>


Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Breast Neoplasms , Allergy and Immunology , CD4-Positive T-Lymphocytes , Allergy and Immunology , Cell Count , Enzyme-Linked Immunosorbent Assay , Interleukin-2 Receptor alpha Subunit , Allergy and Immunology , T-Lymphocyte Subsets , Allergy and Immunology , Transforming Growth Factor beta , Genetics
12.
Journal of Experimental Hematology ; (6): 798-802, 2004.
Article in Chinese | WPRIM | ID: wpr-347859

ABSTRACT

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.


Subject(s)
Humans , Antigens, CD34 , Allergy and Immunology , Colony-Forming Units Assay , Flow Cytometry , Hematopoietic Stem Cells , Cell Biology , Allergy and Immunology , Immunomagnetic Separation , Methods , Reference Standards , Neoplasms , Blood
13.
Chinese Journal of Oncology ; (12): 333-336, 2004.
Article in Chinese | WPRIM | ID: wpr-271020

ABSTRACT

<p><b>OBJECTIVE</b>To investigate whether dendritic cells pulsed with whole tumor lysates (WTL) could in vitro elicit antitumor T cell responses in patients with non-small-cell lung cancer (NSCLC).</p><p><b>METHODS</b>Monocyte-derived immature DCs (imDCs) generated in the presence of human recombinant granulocyte-macrophage colony stimulating factor and interleukin-4 from peripheral blood mononuclear cell of NSCLC patients, and then were induced to mature by pulsing autologous WTL (DCs/WTL) or by the addition of TNF-alpha(TNF/DCs). FACS and MLR assay were used to monitor their phenotypic changes and capacity to stimulate allogeneic and autologous T cell proliferation. DCs/WTL activated with TNF-alpha (* DCs/WTL) were cocultured in vitro with autologous T cells for eliciting antitumor CTLs. T cell mediated antitumor responses were measured by IFN-gamma enzyme-linked immunospot (ELISPOT) assay for WTL-specific IFN-gamma releasing T cells and by lactate dehydrogenase release (LDH) assay for lysis of autologous tumor cells, respectively.</p><p><b>RESULTS</b>When monocytes-derived imDCs from the patients with NSCLC (n = 10) were pulsed with autologous WTL for a day at 30 microg total protein of WTL per 10(6) DCs/ml, this led to up-regulation of CD1a, CD83 and CD86 as well as HLA-DR, and also led to marked stimulation of allogeneic T cell proliferating activity, which was comparable to that of TNF/DCs. However, their capacity of stimulating autologous T cell proliferation in vitro was significantly more potent than those of TNF/DCs (P < 0.05). The numbers of WTL-specific IFN-gamma releasing T cells in 1/3 cultures after one week exposure to * DCs/WTL was increased significantly compared with those pulsing with TNF/DCs plus IL-2 or IL-2 alone (P = 0.05). T cells derived by priming of non-adherent PBMCs with * DCs/WTL after 14 days in vitro stimulation were significantly more responsive to autologous tumor cells compared with LAK (n = 3, P < 0.05), but its cytotoxicity against K562 cells was also comparable to LAK cells.</p><p><b>CONCLUSION</b>Monocyte-derived DCs from NSCLC patients could serve as functional APC. The * DCs/WTL may effectively elicit T cell-mediated antitumor response in vitro and enhance NK killing activity.</p>


Subject(s)
Humans , Antigens, CD1 , Metabolism , Carcinoma, Non-Small-Cell Lung , Allergy and Immunology , Cell Culture Techniques , Cytotoxicity, Immunologic , Dendritic Cells , Allergy and Immunology , HLA-DR Antigens , Metabolism , Interferon-gamma , Bodily Secretions , K562 Cells , Killer Cells, Lymphokine-Activated , Allergy and Immunology , Leukocytes, Mononuclear , Allergy and Immunology , Pathology , Lung Neoplasms , Allergy and Immunology , Lymphocyte Culture Test, Mixed , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Pathology , Tumor Necrosis Factor-alpha , Pharmacology
14.
Chinese Journal of Biotechnology ; (12): 750-753, 2003.
Article in Chinese | WPRIM | ID: wpr-249995

ABSTRACT

Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.


Subject(s)
Humans , Cell Line, Tumor , Cell Survival , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enterotoxins , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Inclusion Bodies , Genetics , Metabolism , Melanoma , Drug Therapy , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Metabolism , Pharmacology , Therapeutic Uses , Single-Chain Antibodies , Genetics , Metabolism
15.
Chinese Journal of Hematology ; (12): 632-635, 2003.
Article in Chinese | WPRIM | ID: wpr-354813

ABSTRACT

<p><b>OBJECTIVE</b>To explore the specific cytotoxic T lymphocyte (CTL) induced by dendritic cells (DC), which were transfected by the plasmid pC53-SN3 encoding p53 gene.</p><p><b>METHODS</b>DC derived from HLA-A2(+) mononuclear cells of the 24-lung cancer patients was transfected with the plasmid pC53-SN3 by lipofectamine and then co-cultured with auto-unpurified T cells to induce potent CTL (T-pC53-SN3). The cytolysis of specific CTL against Calu-6, a HLA-A2(+) human lung cancer cell line, was measured by using lactate dehydrogenase (LDH) releasing assay.</p><p><b>RESULTS</b>The expression of CD(1a) and CD(83), the correlative markers of DC, increased apparently after transfected with plasmid pC53-SN3, the expression rate was (5.45 +/- 0.89)% and (3.26 +/- 0.47)% versus (52.15 +/- 11.56)% and (25.78 +/- 12.35)%. CD(14) decreased apparently, but other DC correlative markers of CD(1a), CD(40), CD(86), and HLA-DR remained almost the same as that before transfection. Compared with T-IL-2, the CTL derived from PBMNC stimulated by IL-2 (100 U/ml), the cytolytic activity of T-pC53-SN3 against Calu-6 cell line showed a significant increase, but cytolytic activity was 56.79 +/- 15.67 and 39.33 +/- 9.88, respectively, when effect cells: target cells was 10:1. The expression of the CD(8), CD(69), and CD(45)RO/CD(8) of T-pC53-SN3 cells increased significantly, but that of CD(3), CD(4), CD(86), ect, was not significantly different from those of T-pCMV-neo.</p><p><b>CONCLUSIONS</b>It showed that DC transfected by p53 gene could induce potent HLA-A(2) restrictive CTL to kill tumor cell efficiently.</p>


Subject(s)
Humans , Antigens, CD , B7-2 Antigen , CD40 Antigens , Cell Line, Tumor , Allergy and Immunology , Coculture Techniques , Cytotoxicity, Immunologic , Allergy and Immunology , Dendritic Cells , Allergy and Immunology , Metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Pharmacology , Interleukin-4 , Pharmacology , Membrane Glycoproteins , T-Lymphocytes , Allergy and Immunology , Tumor Suppressor Protein p53 , Genetics , Physiology
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