| BackgroudThe cancer has become a major killer of the human, and its mortality rate is only second to the cardiovascular disease, and has the upward trend. At present the chemicals not only has a significant effect, but also the toxic side effects. Just as long-term application is easy to produce drug resistance, etc. so people are eager to find a drug with good efficacy and less toxic side effects, and then traditional Chinese medicine in this area shows a clear advantage.The cantharis is the dry worm of Mylabris phalerata Pallas and Mylabris cichorii Linnaeus. The nature of it is cold, and its taste is pungent and high poisonous.Mylabris, a chinese drug, the dried body of Mylabris phalerata Pallas or Mylabris cichorii Linnaeus.It is pungent in taste and hot in nature and toxic, acts on the Liver, Stomach, and Kidney Meridians and has the effects of eliminating blood stasis and masses, combating poisons and cauterizing sores. Indications:massintheabdomen, cancer, chronictinea, scrofula, vegetatio n, abscess difficult to burst, malignant sore and slough. Cantharidin (cantharidin, CA) from the cantharis is the main component of anti-cancer. It has a certain effect on primary liver cancer, breast cancer, gastrointestinal tumors, but the gastrointestinal toxicity has limit its clinical application. Norcantharidin (norcantharidin, NCTD) is the synthesis without 1,2-methyltransferase, based on the chemical results of the cantharidin, not only has significant anti-cancer effect and less side effects, but also can increase the peripheral blood white blood cell, protect the liver cells, and regulate the immune function and so on. Norcantharidin is used Clinically for the treatment of breast cancer, esophageal cancer and gastric cancer. Given the high incidence of liver cancer and the important role of NCTD in the cancer therapy,the research has studied the cell growth inhibition and induction of cell apoptosis of liver cancer H22 and colon cancer Caco2, and anti-liver cancer H22 cells in immunological mechanism was discussed.Objective1. Familiar with and establish a stable method of experimental oncology2. To observe how the norcantharidind to affect the proliferation of tumor3. To observe how the norcantharidin to affect the immune function and bone marrow stem cell proliferationMethods1. The test of hepatoma H22 cell proliferation inhibitionTO set up four concentrations of cells,3×105/ml,2×105个/ml,1×105个/ml,8×104/ml, and be cultured in polystyrene 96-well microtiter plates. H22 cell growth curve will be mapped, to determine the most good delivery time and cell concentration.To prepare 7.5μmol/L,15μmol/L,30μmol/L,60μmol/L,120μmol/L,240μmol/L, 480μmol/L seven concentration gradient solution of NCTD, to effect H22 cells at different time points, and then to use MTT to detect the growth rate of inhibition of H22 cells, and to observe the cell morphology under light microscope.2. To make H22 tumor-bearing mice model and research NCTD's anti-tumor effect2.1 ModelingTo recovery H22 cells at 37℃,and to adjust the cell density to 5×109 /L.0.2ml cell suspension is Injected into the abdominal cavity of each mouse. Around 7d later, the mouse's belly is filled with ascites cells. It can be used for subcutaneous tumors in mice. To collect ascites under sterile conditions in H22 mice and to dilute it with 1640. To centrifuge cells (1000r/min,5min). They were washed 2 times, and then to adjust the density of H22 cell by 1×109/L.2.2 Animal group and administration72 NIH mice, are fed adaptively for 4d and divided randomly into normal control group, model group, cyclophosphamide group, norcantharidin to a low, medium and high dose group, doses of 1,2,3 mg·kg-1·d-1, a total of 6 groups and each group has 12 rats (male and female). In addition to the normal control group,0.2mL of H22 cells were injected subcutaneously in the right forelimb armpit of the other5groups of mice. After inoculation for 24h, cyclophosphamide group and norcantharidin of a low, medium and high dose groups are injected intraperitoneally 50mg/kg of cyclophosphamide for the first time, and then for the second time on the 8th day. The normal group, model group, cyclophosphamide group are fed with the saline by 0.2ml/10g for 14d; the norcantharidin of low, medium and high dose group are injected with norcantharidin forl4d. To determine the growth of H22 tumor by vernier caliper.2.3 To study the tumor in PharmacodynamicsThe experimental mice are killed on thel5th day, and peel the tumor to weigh them. Inhibitory rate was calculated:Pinhibtion (%)= (mmodel group-mexperimental group) /mmodel group×100%.3. Mechanism of antitumor of antitumor immune parameters related to the immune function.3.1 measuring immune organsThe experimental mice are killed on thel5th day, spleen and thymus respectively to weigh them, spleen index=spleen weight (mg)/body weight (g), thymus index=thymus weight (mg)/body weight (g). 3.2 The lymphocyte transformation test to determine the lymphocyte stimulation index of spleen.3.3 To test the change of proportion of spleen T cell subsets CD3+, CD4+, CD8+ and NK cell activity by flow cytometry.3.4 To test the level of IL-2, TNF-αin the serum of mice by ELISA.3.5 To test the changes of bone marrow nucleated cell apoptosis and cell cycle by flow cytometry.4. The test of colon cancer cell Caco2 proliferation inhibition and apoptosis in vitro.TO set up four concentrations of cells,1.2×105/ml,1×105个/ml,8×104个/ml,5×104/ml, and be cultured in polystyrene 96-well microtiter plates Caco2 cell growth curve will be mapped, to determine the most good delivery time and cell concentration. To prepare 7.5μmol/L,15μmol/L,30μmol/L, 60μmol/L,120μmol/L,240μmol/L,480μmol/L seven concentration gradient solution of NCTD, to effect Caco2 cells at different time points, and then to use MTT to detect the growth rate of inhibition of Caco2 cells, and to observe the cell morphology under light microscope. And Caco2 cell cycle and apoptosis rate are measured by flow cytometry.Results1 NCTD inhibite of proliferation of H22 hepatoma cells in vitro.7.5 u mol/L,15μmol/L,30μmol/L,60μmol/L,120μmol/L,240μmol/L,480μm ol/L, seven different concentrations of NCTD, affected H22 tumor cells at different time points. We found the effect of NCTD increased with the concentration of NCTD within 72h.There is a significant time-effect and dose-effect relationship, and IC50 is 60μmol/L of the NCTD at the point of 36h. Before being cultruing with norcantharidin, H22 were observed under the inverted microscope in good cell growth, cell morphology and no significant difference in the number. After adding the appropriate concentration of norcantharidin to the wells, cells were observed. We can see apoptotic bodies gradually increased damaging membrane, and pieces like cells gradually more though microscope.2 Modeling H22 tumor-bearing mice and studying anti-tumor effect of NCTD.NCTD significantly inhibited H22, and the inhibition rate and a certain dose was positively correlated. Norcantharidin combined with the high dose CTX, has the strongest tumor inhibition, it's tumor weight is the minimum and inhibitory rate was 65.11%, so the difference was statistically significant, compared with the CTX group (P<0.05).3. The effct NCTD on immune function of mice3.1 The effct of NCTD on immune organs.From the thymus index results, the low dose of norcantharidin group is 3.19±1.24 and 2.38±0.61 of cyclophosphamide alone group.the result is statistically significant compared. From the spleen index results, medium dose of norcantharidin is to a ratio of 9.78±4.24, compared with cyclophosphamide alone group,5.42±1.11, and the effect is significant (P< 0.01). So norcantharidin can significantly inhibite H22, and there is increased thymus, spleen index effect.3.2 The effct of NCTD on lymphocyte proliferation.Compared with the CTX group 1.97±1.63, medium and high doses of NCTD group 5.35±0.83,7.94±3.87 are significantly different (P<0.05, P<0.01), but low dose group 3.18±1.07 has no significant difference; compared with the model group 2.73±0.97, high-dose of NCTD group 7.94±3.87 has significant difference (P<0.01). So norcantharidin can increase the immune suppression of lymphocyte function in mice.3.3 The effct of NCTD on spleen T cell subsets.By flow detector showed that CTX group and high-dose NCTD with CTX group, compared with normal groupd and model group, can inhibit the immune spleen cells (P<0.01 or P<0.05); compared with the CTX group, of low-dose NCTD with CTX group,CD3+ is 11.4%(P<0.01), CD4+ accounts for 7.3% (P<0.05), CD8+ is 2.88%(P<0.05), and is statistically significant, but there is no difference with the normal and model group; compared with the normal group, medium-dose NCTD can affects CD3+ T cells in the spleen (P<0.05), but the other is not obvious.3.4 The effct of NCTD on NK cell activity in mice.High-dose NCTD 29.17±1.4%, while the normal group 32.7±2% has no significant difference, indicating that high doses of NCTD can significantly improve the H22 tumor-bearing mice NK cell activity. And there is also statistically significant compared with model group and CTX group (P<0.01).3.5 The effct of NCTD on serum IL-2 and TNF-α.From the ELISA assay results, we can see compared with the control group 23.6±2.28 pg·ml-1, the medium-dose and high-dose NCTD with CTX groups 37.08±0.23 pg·ml-1,47.83±1.75 pg·ml-1 were significant differences (P<0.01). NCTD can effectively stimulate the secretion of IL-2 in H22 tumor bearing mice; and compared with CTX group 15.71±3.06 pg·ml-1, three doses of NCTD groups 28.09±0.45pg·ml-1,37.08±0.23pg·ml-1,47.83±1.75pg-ml-1 were significant (P< 0.05, P<0.01). NCTD can effectively stimulate the secretion of TNF-αin H22 tumor bearing mice. Compared with the CTX groupll.87±2.73 pg·ml-1, medium-dose and high-dose NCTD with CTX groups 19.97±0.94 pg·ml-1,45.1±0.52pg-ml-1 were statistically significant (P<0.05, P<0.01).4. The effct of NCTD on mouse bone marrow cells.The results show that compared with the CTX, low-dose NCTD with CTX dose not affect of bone marrow cells, but medium-dose and high-dose NCTD with CTX can increased the apoptosis rate of bone marrow cells (P<0.01); CTX group shows that it will prevent the bone marrow cells in the G2/M phase, the low dose of NCTD with CTX does not affect bone marrow cells, medium-dose and high-dose NCTD with CTX significantly reduced the ratio of this cell cycle (P<0.05, P<0.01), and the cell ratio of G1/G0 phase has no significant difference.5. The effect of NCTD on colon cancer cell Caco2 proliferation inhibition and apoptosis in vitro7.5μmol/L,15μmol/L,30μmol/L,60μmol/L,120μmol/L,240μmol/L,480μm ol/Lseven different concentrations of NCTD affected Caco2 tumor cells at different time points. We found the effect of NCTD increased with the concentration of NCTD within 48h.There is a significanttime-effect anddose-effectrelationship. Incubated with norepinephrine before cantharidin, Before being cultruing with norcantharidin, Caco2 were observed under the inverted microscope in good cell growth, cell morphology and no significant difference in the number. After adding the appropriate concentration of norcantharidin to the wells, cells were observed.We can see apoptotic bodies gradually increased damaging membrane, and pieces like cells gradually more though microscope. The apoptosis rate of NCTD in 60μmol/L is the highest within 48h, and then the apoptosis rate decreased with the increasing of NCTD's concentration, but the cells of G0/G1 increased.Conclusions1. NCTD can inhibit the proliferation of H22 cells, and there is a significant time-dose-effect relationship;2. Nrocantharidin significantly inhibit H22, and the inhibition rate and the certain dose is positively correlated. Its mechanism may improve immunityand enhance cellular immune function.3. NCTD can inhibit the proliferation of Caco2 cells, and there is a significant time-dose-effect relationship, and affect both the apoptosis rate of cells and cell cycle;... |
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