| Objective :To further investigate the antitumor responses of a novel human bone marrow stromal cell-derived growth inhibitor(BDGI), and apply the recombination plasmid pcDNA3.1-BDGI to 4T1 murine mammary carcinoma model by intratumoral injection and evaluate its antitumor efficacy and possible antitumor mechanisms in tumor bearingmice.Methods :1. Animal experiment54 mice were inoculated s.c. with 1×10~6 4T1 mammary tumor cells to establish experimental animal model of breast cancer. After 24 hours of injection,mice were randomly divided into 6 groups: the control group, theblank plasmid group, the treatment group I, the treatment group II, the CTX group, the Joint Unit group. Different doses of the drug were injected s.c. in mice and treated for three times (the interval of 3 days). Surveied bearing mice health, living conditions, the growth of tumors. On the seven day after the last treatment, executed from cervical of mice (3/group) for the spleen lymphocytes, MTT measured NK cytotoxicity assay. Collect the supernatant, store at -20°C, to be detected cytokines; Weighing in tumor tissue from 10% formalin-fixed, pending pathological observation; The lungs, 10% formalin -fixed, pending pathology observed tumor metastasis; the remaining miceretained for survival observation. 2. NK cytotoxicity assayNK cytotoxicity assay was measured by the MTT colorimetry assay.Executed the mice in sterile conditions and removed the spleen, after 120 Mesh filters remove adipose tissue, from cell suspension system, breaking red. adjust its concentration of 2×10~6/ml, directly used as effector cells. YAC-1 as target cells in the concentration of 2×10~5/ml, MTT measured NK cell samples. In briefly, YAC-1 cells (2×10~5/ml) as target cells were seeded in 96-well plastic plate. Spleen cells were prepared from the mice as effector cells and simultaneously seeded with YAC-1 (2×10~6/ml) at 10:1 ratios of effector to target (E:T) and total of volume was 200μl. After the plate was incubated at 37°C in 5% CO2 for 8 hours, the supernatant was discarded, and then each well was added with10μl MTT for additional 4 hours incubation. The supernatants were discarded and 150μl dimethylsulfoxide was added and shaken for a minute, then optical densities were measured with the plate reader (Bio-rad) at OD 570.3. pathology observed In tumor tissue, lung tissue followed after 10% formalin-fixed, decalcified EDTA, ethanol dehydration gradient, conventionalembedded and sectioned at HE staining and microscopy observation of histopathologic changes, as compared to the differences between the groups. 4. Dectection of Cytokines Cytokines collection of T lymphocytes were cultured medium. IL-2, TNF-α and IFN-γ were dectected in accordance with the EIA requirements kit brochures. Put culture supernatant or the analyte concentration of the standard 100 ul/well, 37°C for 120 minutes, wash five times, in each well to work with the first antibody 50ul. 37°C for 60 minutes, wash five times, each well ELISA antibody fluid 100ul, 37°C for 60 minutes, wash five times, each well to work with the substrate, while the reaction 100 ul, 37°C five minutes, 50 ul per well to terminate the reaction, 490 nm absorbance detection wavelength, drawing standard curve calculation results.Results :1. Pathological change of the tumor and the lungTumor tissue:The tumor cells vary in size and shape, there was much more spread of nuclear debris, markedly abnormal cells. Compare with the control group, the blank plasmid group, each drug treatment group was a certain degree of inflammatory cells(neutrophils,lymphocytes) infiltration accompanied by some of the tumor necrosis, but the difference among the groups was not significant. Lung tissue:The control group, the blank plasmid group, the treatment group I and the treatment group II was observed in lung metastases. Tumor cell sizes, the shape of a melon or oats, was part of the cords arranged between sinusoids,accompanied by a certain degree of inflammatory cell infiltration. The CTX group, the joint group appeared no metastases, tumor metastasis were significantly inhibited, the control group, the blank plasmid group, the treatment group I, the treatment group II, the CTX group, the Joint Unitgroup.2. Improvement activities of NK in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group the treated mice NK cytotoxic activity was significantly increased (P<0.05); With the blank plasmid group, NK cytotoxic activity of the other treatment groups were significantly increased (P<0.05); Compared with the CTX group, the CTX group NK cytotoxic activity was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group, the Joint group , the treatment group II NK cytotoxic activity were significantly increased (P<0.05).3. Level of cytokine3.1 Improvement activities IFN-γ level in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group IFN-γ level was no significant difference(P>0.05), other groups IFN-γ level were significantly increased (P<0.05); Compared with the blank plasmid group, other groups IFN-γ level were significantly increased (P<0.05); Compared with the CTX group, the CTX group IFN-γ level was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group , the Joint group, the treatment group II IFN-γ level weresignificantly increased (P<0.05).3.2 Improvement activities IL-2 level in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group was no significant difference, while other groups IL-2 level were significantly increased (P<0.05); Compared with the blank plasmid group, other groups IL-2 level were significantly increased (P<0.05); Compared with the CTX group,the CTX group IL-2 level was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group, the Joint group , the treatment group II IL-2 level were significantly increased(P<0.05).3.3 Improvement activities TNF-αlevel in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group TNF-αlevel was no significant difference, while other groups TNF-αlevel were significantly increased (P<0.05); Compared with the blank plasmid group, other groups TNF-αlevel were significantly increased (P<0.05); Compared with the CTX group, the CTX group TNF-αlevel was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group, the Joint group, the treatment group II TNF-αlevel were significantly increased (P<0.05).Conclusions :1. In 4T1 mouse model of breast cancer, the companied BDGI with CTX treatment, improvement of BDGI dose treatment, can inhibit tumor growth.The treatment group II and the Joint group were extend the survival of mice.2. company BDGI with CTX or raise BDGI dose can increase splenic NK cytotoxic activity, and induce more Th1 cytokines (IL-2, IFN-γ, the secretion of TNF-α, induce specific and non-specific anti-tumor immune response.3. Company BDGI with CTX can effectively inhibit tumor metastasis to the lung.4. Combined therapy and high-dose treatment of tumor-bearing mice can induce more inflammatory cell infiltration, tumor tissue necrosis, However, the difference between the groups was not significant.5. The BDGI treatment of breast cancer is a certain dose-dependent relationship. These results suggest BDGI may have potential application of cancer gene therapy. Maybe explore a new cancer treatment method and new way to provide new ideas.
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