| Colon cancer is the common malignant tumors of alimentary tract, with the improvement of living standard and living habits, in recent years the incidence of colon cancer presenting the obvious trend of rising. Current treatment of advanced colon cancer is still adhere to put, chemotherapy. But in the course of treatment, drug side effect is great, the life quality of the patients is very low. Current clinical application of drugs, more successful chemotherapy drugs are tumor cell mitosis inhibitors such as paclitaxel. Tao after a lot of study found, tumor apoptosis may be associated with M block (M-arrest) and M (M-slippage) mechanism of slip. The research shows that, when the cells using a drug arrest in the M phase, adding another drug can promote apoptosis of tumor cells.Traditional Chinese medicine for tumor, especially in advanced tumor therapeutic effect is remarkable, can significantly improve cancer survival and reducing, chemotherapy drug side reaction. A large number of studies show that traditional Chinese medicine effective constituents, some can effectively inhibit tumor cell proliferation, promote their apoptosis. Evodiamine is so far reported having resistance to tumor growth, directly induce apoptosis in tumor cells, inhibition of tumor invasion and metastasis is one of the effective constituents. Our previous research also confirmed that evodiamine in G2/M phase induced apoptosis of tumor cells, and this M block (M-arrest) and M slip (M-slippage) tumor apoptosis mechanisms coincide.The first part of the experiment research of evodiamine combined with CDKlinhibitor R0-3306or Indigo jade red on rat colon cancer CT26 cells synergistic effect, in the M block (M-arrest) and tumor apoptosis mechanism foundation, to investigate the occurrence of M block (M-arrest) and M slip (M-slippage) time point and the sequential administration will the M slip (M-slippage) rises to promote action, in response to disprove evodiamine combined with CDKlinhibitor on rat or human colon synergistic cytotoxicity and M block (M-arrest) and M slip (M-slippage) tumor apoptosis mechanisms coincide. And in the same experimental conditions and Experimental Methods Tentative verification of paclitaxel induced after joining M-arrest, CDKlinhibitors, aimed at elucidating the paclitaxel induced tumor apoptosis mechanism and M block (M-arrest) and M slip (M-slippage) tumor apoptosis mechanism. For the combined use of Chinese and Western medicine resisting tumor to do basic research.Research methods:2.1. lusing flow cytometry (PI single dye) method confirm evodiamine induces CT26cell cycle arrest onset time points were set up:control group, synchronization control group, control groupl8hours,18 hours of EV02mg/L group,18 hours of EV04mg/L group, control group, the 2121 hours EV02mg/L group,21 hours of EV04mg/L group, control group24hours,24 hours of EV02mg/L group,24 hours of EV04mg/L group, control group27hours,27 hours of EV02mg/L group,27 hours of EV04mg/ L group, control group30hour,30 hours EV02mg/L group,30 hours of EV04mg /L group, after collecting cells, after PI fixation of single dye, were detected by flow cytometry.2.1.2WESTERNBLOT method for confirmation of evodiamine induces CT26ce11 cycle arrest appear time points:as the packet, a collection of protein, were detected in3-actin, Bax, CyclinE expression.2.1.3using flow cytometry (PI single dye) detection of evodiamine combined with CDKlinhibitor RO-3306 on rat colon cancer CT26cell cycle and apoptosis rate effects, observed changes in cell morphology. Respectively setting controls, EV02mg/L group, EVO group,4mg/L EV02mg /L and RO-330615mg/L combined drug group, EV04mg/L and RO-330615mg /L combined drug group, EV02mg/L24 hours after joined the RO-330615mg /L6,4mg/EVO group effect of L24 hours after the accession to the RO-330615mg/L RO-330615mg/6 hour group, L group, RO-33061ast 6 hours of 15mg/L group, the action time of the drug in each group for 30 hours, after collecting the cells, after PI fixation of single dye, were detected by flow cytometry.2.1.4 WESTERNBLOT method for determination of evodiamine combined with CDK1inhibitor RO-3306 on rat colon cancer CT26cell cycle and apoptosis rate influence. As a group, a collection of protein, were detected in3-actin, Bax, CyclinE expression.2.1.5using flow cytometry (PI single dye) detection of evodiamine joint indigo jade red on rat colon cancer CT26cell cycle and apoptosis rate effects, observed changes in cell morphology. Respectively setting controls, EV02mg/L group, EVO group,4mg/L EV02mg/L and indigo jade red 20mg/L combined drug group, EV04mg/L and indigo jade red 20mg /L combined drug group, EV02mg/L24 hours after adding indigo jade red 20mg/L6hour group, EV04mg/L24 hours after adding indigo jade red 20mg /L function6 hour group, indigo jade red 20mg/L role last6 hour group, indigo jade red 20mg/L group, the total of each drug for30 hours, after collecting cells, after PI fixation of single dye, flow cytometry.2.1.6WESTERNBLOT method for determination of evodiamine and indigo jade red on rat colon cancer CT26cell cycle and apoptosis rate influence. As a group, a collection of protein, were detected in3-actin, Bax, CyclinE expression.2.1.7using flow cytometry (PI single dye) detection of paclitaxel combined with CDK1 inhibitor RO-3306 on rat colon cancer CT26cell cycle and apoptosis rate effects, observed changes in cell morphology. Respectively setting controls, paclitaxel2mg/L group, RO-3306group,15mg/L andl5mg/L RO-3306paclitaxel combined with drug, taxol 2mg/L after 24 hours to join the RO-330615mg/L6hour group, RO-330615mg/L role last 6hour group, total time each drug for30 hours, after collecting the cells, after PI fixation of single dye, were detected by flow cytometry.2.1.8WESTERNBLOT method for the detection of paclitaxel combined with CDKlinhibitor RO-3306 on rat colon cancer CT26cell cycle and apoptosis rate influence. As a group, a collection of protein, were detected in3-actin, Bax, CyclinE expression.2.1.9using flow cytometry (PI single dye) detection of paclitaxel combined with indigo jade red on rat colon cancer CT26cell cycle and apoptosis rate effects, observed changes in cell morphology. Respectively setting controls, paclitaxel2mg/L group, indigo jade red 20mg/L group, paclitaxel and indigo jade red 20mg/L combined drug, taxol 2mg/L after 24 hours to join the indigo jade red 20mg/L6hour group, indigo jade red 20mg/L role last 6hours group, the role of total time each drug for30 hours, after collecting cells, after PI fixation of single dye, were detected by flow cytometry.2.1.10WESTERNBLOT method for the detection of paclitaxel combined with indigo jade red on rat colon cancer CT26cell cycle and apoptosis rate influence. As a group, a collection of protein, were detected in3-actin, Bax, CyclinE expression.Conclusion1, evodiamine induces apoptosis in colon cancer cells apoptosis mechanism of CT26with M-arrest and M-slippage apoptosis mechanism. But when joining CDK1 inhibitor R03306, because of many factors, the mechanisms have not been clearly, presumably in some concentrations, evodiamine and R03306antagonist, some concentrations appeared synergistic.2, paclitaxel and CDKlinhibitor RO-3306sequential or combination application did not show a synergistic effect, and even more showed antagonistic. It has great reference significance for clinical medication, clinical application of paclitaxel with CDKI inhibitors in the treatment of tumors should be careful!3, evodiamine joint indigo jade red role for CT26 cells, under certain conditions, may show synergy effect, may also exhibit both antagonistic effect. For further study.4, paclitaxel and indigo jade red combined or sequential medication effect on colon cancer cells CT26showed antagonistic relationship. This prompts in clinical medication, paclitaxel and indigo (mainly indigo jade red) can not be combined application. This is for the combined use of Chinese and Western medicine treatment of tumor presents a taboo. |
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