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The background tumor has gradually become one of the major human killer mortality after cardiovascular and cerebrovascular diseases, but also showed an upward trend. Anti-tumor effects of chemical drugs exact a significant effect, but the toxic side effects, long-term application is easy to produce drug resistance, etc., so people are eager to find better efficacy and toxicity of drugs, traditional Chinese medicine in this regard shows a distinct advantage. Of cantharidin (Mylabris) Chinese medicine name. Dried worms, daphne Southern Youth Division insects cantharidin or yellow and black small cantharidin of spicy, hot, big drug, the liver, stomach, kidney, the efficacy Poxue Xiaozheng attack poison eclipse sores, foam cold for a long time . The Zhengjia used for cancer, the Jinian stubborn ringworm, M. scrofulaceum, wart, ulcer does not collapse, malignant sore dead muscle. Modern pharmacology studies suggest that the main component of cantharidin cantharidin anticancer, have a certain effect on primary liver cancer, breast cancer, gastrointestinal cancer, but because of toxic effects on the gastrointestinal tract, such as limiting its clinical Application. Go norcantharidin (norcantharidin, NCTD) according to the results of chemical cantharidin, go to the synthesis of 1,2-methyltransferase, not only has significant anti-cancer effect and less side effects, also has Shengbai, protect liver cells regulate immune function. Mainly used in the clinical treatment of breast cancer, esophageal cancer, gastric cancer, and in view of the high incidence of the tumor and NCTD an important role in cancer therapy, this study examines the NCTD H22 cells and colon adenocarcinoma Caco2 cell growth inhibition NCTD and the induction of apoptosis, and anti-H22 cells immunological mechanism was discussed. Purpose of a familiar and to establish a stable of experimental tumors science research methods 2 observation go norcantharidin on tumor proliferation of 3 observation go norcantharidin nuclear cell proliferation impact method of immune function and bone marrow 1 hepatoma H22 cells in vitro proliferation inhibition experiments MTT assay NCTD H22 cells growth inhibition rate. Set 3 × 105 / ml, 2 × 105 / m1, 1 × 105 / m1, 8 × 104 / ml four concentrations of cell suspension was inoculated 96-well plate, drawing H22 cell growth curve, determine the best time of administration and the cell concentration. Preparation of 7.5μmol / L, 15μmol / L, 30μmol / L, 60μmol / L, 120μmol / L, 240μmol / L, 480μmol / L of seven concentration gradient NCTD solution, the role of H22 cells different time points using the MTT assay technology statistics the NCTD growth inhibition rate, cell morphology was observed under an optical microscope. 2 H22 anti-tumor effect of tumor-bearing mouse model of production and NCTD study the production of the 2.1 H22 tumor-bearing mouse model: 37 ℃ recovery H22 cells, the cell density was adjusted to 5 x 109 / L, 0.2ml / only sterile injection NIH mice by intraperitoneal inoculation passages about vaccination 7d, the mouse abdominal distension, inoculated subcutaneously transplanted tumors in mice can be used. H22 ascites tumor mouse ascites, diluted 1640 drawn under sterile conditions, centrifuged (1000r/min, 5min) cells were washed twice, adjusting H22 cell density of 1 × 109 / L, in addition to the normal control group the remaining five groups in the right forelimb armpit subcutaneous injection of H22 cell suspension 0.2mL. 2.2 Animal grouping and method of administration: NIH mice 72 4d, were randomly divided into normal control group, model group, cyclophosphamide group, go norcantharidin low, medium and high dose groups, dose, respectively, adaptability keeping , 2, Smg · kg-1 · d-1, a total of six groups (n = 12) (male and female). Inoculated 24h after cyclophosphamide group and go to norcantharidin low, medium and high dose groups intraperitoneal injection of cyclophosphamide 50mg/kg chemotherapy first eight days of chemotherapy second; normal group, model group, cyclophosphamide group started 0.2ml/10g respectively gavage to saline continuous administration 14d; go norcantharidin low, the high-dose group Intraperitoneal injection of cantharidin, continuous administration of 14d; H22 tumor growth is determined by a vernier caliper. 2.3 tumor and immune organ measurement experiment Rule 15d mice were sacrificed, peel the tumor, spleen, thymus, respectively, said the quality of inhibitory rate was calculated: P tumor inhibition (%) = (m model group-m experimental group) / m model group × 100%. Spleen index = thymus quality (mg) / body weight (g), thymus index = thymus weight (mg) / body weight (g). Determination of immune function indicators lymphocyte transformation test determination of mouse spleen lymphocytes stimulation index 3.1. 3.2 was measured by flow cytometry method splenic T cell subsets CD3, CD4, CD8 in the change in mouse bone marrow nuclear apoptosis and cell cycle changes, and NK cell killing activity changes. 3.3 using the ELISA, the experimental serum IL-2, TNF-α content by line determination. 4 NCTD colon cancer Caco2 cells in vitro inhibition of proliferation and apoptosis in experimental design 1.2 × 105 / ml, 1 × 105 / ml, 8 x 104 / m1, 5 x 104 / ml four concentrations of cell suspension inoculated 96-well plate, drawing the Caco2 cell growth curve, to determine the optimal dosing time and cell concentration. 7.5μmol / L, 15μmol / L, 30μmol / L, 60μmol / L, 120μmol / L, 240μmol / L, 480μmol / L solution of seven concentration gradient NCTD role Caco2 cells at different time points after the preparation, the use of the MTT assay technologies statistics NCTD growth inhibition rate, cell morphology was observed under an optical microscope, and the Caco2 cell growth inhibition rate and apoptosis rate was determined using flow cytometry techniques. Results 1 NCTD H22 cells in vitro proliferation inhibitory effects of seven different concentrations (7.5μmol / L, 15μmol / L, 30μmol / L, 60 μmol / L, 120 μmol / L, 240 μmol / L, 480μmol / L,), NCTD different point in time the role of H22 tumor cells and found that the NCTD within a certain amount of time and concentration range, the inhibitory effect of H22 cells increased with the increased concentration of NCTD and prolonged duration of action, with the time - and dose - effect relationship, IC50 as the H22 cells 36h 60μmol / L NCTD role. Go norcantharidin incubation, the cells grew well observed under an inverted microscope, cell morphology with no significant difference in the number, add the appropriate concentration go the 6 norcantharidin microscope cell concentration, with time, measurable increase in endoscopic apoptotic bodies appeared and gradually increased, membrane damage, pieces like cells gradually more. 2 NCTD to norcantharidin anti-tumor effect of tumor-bearing mice model of H22 significantly inhibited, and was certainly a positive correlation between inhibition rate of dose. Go to the most high-dose CTX inhibitory effect of the combination group norcantharidin, tumor weight minimum inhibition rate was 65.11%, compared with the CTX, the difference was statistically significant (P lt; 0.05). From the results of the thymus index, low dose to norcantharidin 3.19 ± 1.24 with a mere cyclophosphamide compared to 2.38 ± 0.61 with statistical significance. 3.1 pairs of 3.NCTD tumor-bearing mice immune function of immune organs: spleen index results in dose to norcantharidin 9.78 ± 4.24 than the simple effect of cyclophosphamide 5.42 ± 1.11 significantly (P lt; 0.01). Description of H22 go norcantharidin significantly inhibited, and improve the effect of the thymus, spleen index. Medium and high dose the NCTD group, 5.35 ± 0.83,7.94 ± 3.87, significant difference (P lt; 0.05 3.2 pairs of lymphocyte proliferation, and CTX group compared to 1.97 ± 1.63, P lt; 0.01), low-dose group 3.18 ± 1.07 the difference was not significant; compared with the model group, 2.73 ± 0.97, high dose NCTD group 7.94 ± 3.87, significant difference (P lt; 0.01). Description go the norcantharidin can effectively improve the immunosuppressed mice lymphocyte function. 3.3 splenic T cell subsets by flow detector, the results show that the CTX group and NCTD high-dose combination the CTX group with the normal group, model group, spleen cells immune inhibition (P lt; 0.01 or P lt compared with the CTX group; 0.05);, NCTD low-dose combination the CTX group CD3 11.4 percent), CD4 accounted for 7.3% (P lt; 0.05), CD8 is 2.88% (P lt; 0.05), there are statistically significant, with the normal relatively undifferentiated group, model group; compared with the normal group, middle dose NCTD affect the of spleen CD3 T cells (P lt; 0.05), other significant effect. 3.4 pairs of NK cell activity in the the NCTD high-dose group and 29.17 ± 1.4% with the normal group, 32.7 ± 2% of no significant difference, indicating that the high dose of NCTD can significantly improve the H22 tumor activity of NK cells by CTX group can also be seen with the NCTD high dose group, a statistically significant (P lt; 0.01), compared with the model group, only the NCTD high dose group was statistically significant (P lt; 0.01). 3.5 on mice of serum IL-2 and TNF-α affect the use of the ELISA assay results of NCTD in the high dose group 37.08 ± 0.23pg · ml-1, 47.83 ± 1.75pg · ml-1 with the normal group, 23.6 ± 2.28pg · ml-1 difference significant (P lt; 0.01), the NCTD effective stimulus H22 tumor-bearing mice secrete IL-2, so that the elevated levels of serum IL-2; CTX group was 15.71 ± 3.06pg · ml-1 Compare the NCTD three dose groups 28.09 ± 0.45pg.ml-1, 37.08 ± 0.23pg · ml-1, 47.83 ± 1.75pg.ml-1 were statistically significant (P lt; 0.05, P lt; 0.01). NCTD can effectively stimulate the H22 tumor secretion of TNF-α, serum TNF-α levels increased with the CTX group 11.87 ± 2.73pg.ml-1 compares the the CTX joint NCTD, the high-dose group TNF-α19.97 ± 0.94pg · ml-1, 45.1 ± 0.52pg.ml-1 were statistically significant (P lt; 0.05, P lt; 0.01). 4 of mouse bone marrow nucleated cells affect experimental results show that, compared with the CTX The cytotoxicity of NCTD in low-dose CTX combined nuclear apoptosis rate did not affect bone marrow NCTD in dose and high dose CTX combination can improve bone marrow nucleated apoptosis rate (P lt; 0.01); the CTX group of bone marrow nucleated cell block in the G2 / M phase, CTX the NCTD for that combination of low doses of nucleated bone marrow cells did not affect, in combination, the high doses of NCTD significantly reduce the cycle of the cell ratio (P lt; 0.05, P lt; 0.01), at the G1 / G. Phase of the cell ratio was no significant difference. 5 NCTD colon cancer Caco2 cells in vitro inhibition of proliferation and apoptosis of seven different concentrations (7.5μmol / L, 15 μmol / L, 30μmol / L, 60 μmol / L, 120 μmol / L, 240 μmol / L, 480μmol / L ) the role of the different time points Caco2 tumor cells, and found that NCTD within a certain time and the concentration range, the inhibition of the Caco2 cells is increased with NCTD of concentration and action time, with obvious time - effect NCTD and the dose - response relationship. Go norcantharidin incubation, the cells grew well observed under an inverted microscope, cell morphology with no significant difference in the number, add the appropriate concentration go the 6 norcantharidin microscope cell concentration, with time, measurable increase in endoscopic apoptotic bodies appeared and gradually increased, membrane damage, pieces like cells gradually more. The streaming detection the NCTD role Caco2 cells, respectively, 24h, 48h, and found that with increasing dose, the percentage of apoptotic cells gradually increased, in a certain period of time within a certain concentration range, 60 μmol / L the NCTD role Caco2 cells 48h, apoptosis The rate is the highest, the The apoptosis rate declined When NCTD concentrations continue to rise. Conclusion 1 go H22 tumor cells norcantharidin inhibited NCTD concentration increased and prolonged duration of action, inhibition is more obvious with time - and dose - response relationship; 2 go norcantharidin H22 significantly inhibited tumor cell and tumor inhibition rate was certainly a positive correlation between dose. Its mechanism of action may enhance immune function and improve immunity. 3 go norcantharidin on Caco2 tumor cells significantly inhibited in a certain time and concentration range, NCTD, inhibition of the Caco2 tumor cells enhanced with the increased concentration of NCTD and prolonged duration of action, with a significant The time - and dose - response relationship.
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