| Background and objective: Cancer has become the number one human health hazards of disease, conventional chemotherapy and radiotherapy treatment for cancer is unsatisfactory. When cancer patients receiving radiotherapy and chemotherapy to kill tumor cells at the same time, the majority of anti-tumor drugs lack of choice, so to normal body there are also quite big damage, Which lead to bone marrow suppression, functional gastrointestinal reactions, organizations and liver and kidney damage. Chinese herbal medicine with chemotherapy and radiotherapy can reduce the side effects of cancer and there have been noticeable effects may have to raise. Purely objective partial remission and does not reflect the effect of Chinese medicine. Systemic treatment, improving the symptoms, and improving the quality of life are the characteristics of Chinese medicine in curing cancer. So the natural and biological components of the plant extract effective drug as a new chemical anti-cancer agent, has become the research workers of the most interesting research topic. The purpose of this experiment to investigate the mechanism of aqueous extract of toad skin kill tumor cell out of body. Chinese anti-tumor mechanism is complex, When single Chinese herb compound preparation, contain a variety of ingredients, whose role is not single, but multifaceted. How multilevel and multidisciplinary on the role of traditional Chinese medicine anti-tumor mechanism of objective, quantitative and qualitative research to further reveal its mystery, it needs Chinese medicine science workers to work harder. We can say that confidently, after several thousand years ago, Chinese medicine theory and practice based on experience with theory and modern biotechnology drugs, the effect of Chinese medicine research in anti-tumor and the mechanisms of clinical application will be have great development. Methords:1. Cell culture: The liver tumor cell SMMC-7721were cultured in DMEM media with 10% new-born calf serum,100U/ml penicillinum and streptomycin on the condition of 37oC,5%C02.2. MTT assay: The suspended cells obtained from the exponential phase of growth Cells were incubated into 96~well Plates. Setting up blank group added no hepatoma carcinoma cell. 24hours later, the drug was added into drug group. Negative group was added into culture solution. At the end of 4hours, 8hours, MTT was added, then to test.(1)Cell survival ratio is for rates Y axle, survey time is for X axle to make 4 to 72 hours'cells growth curve.(2)Utilize formula to calculate generation inhibition ratio of 24hours,48 hours and 72 hours.(3)Utilize EXCEL to draw picture of 50% inhibiting concentration of 24 hours, 48hours and 72 hours.3. Colony formation experiment: Collect exponential phase of growth cells, inoculate into 6-wells plates. Every plate is 200 cells. Two ways were used to detect the condition of colony formation. Set up drug group and add different saturation'drug Negative drugs adds to isovolumic culture solution. Detect 48 hours later detected the condition of colony formation.4. Transwell experiment: Drug action to cells 24 hours later, cells inoculated to cave and cultured 24 hours on the condition of 37oC, 5%C02. Then take out of cave, coloretured and deaquated the cave and so on. Slice the membrane of cave in the bottom. Use neutral gummi to mount the membrane. Use light microscope to observe cell population that countersink membrane in every 5 eyesight.Results:1. MTT assay :(1)Proliferation inhibition ratio result: The 24 hours later, when saturation of drug is 0.03125mg/ml,0.0625mg/ml and 0.25mg/ml, the proliferation inhibition ratio is 13%, 20% and 53%.48 hours later, the proliferation inhibition ratio is 22%, 37% and 68%. 48 hours later, the proliferation inhibition ratio is 58%, 75% and 82%.Which compared with the negative control group has notable effect (P<0.05).(2)Growth curve result: Showed the cells of experiment group grew slowly.(3)(IC50)result: In the 24 hours, the value of IC50 is 0.2176mg/ml, in the 48 hours, the value of IC50 is 0.0655mg/ml. In the 72 hours, the value of IC50 is 0.0237mg/ml.2. Colony forming efficiency result: When drug saturation is 0.005 mg/ml, added to cells, the 48 hours later, conventional culture 6 days, the colony forming efficiency is12.4%,conventional culture 5 days, add drug to cells, the 48 hours later, the continue to culture 3 days, colony forming efficiency is14.3%,which compared with the negative control group has notable effect(P<0.01).3. Transwell result: When drug saturation is 0.005 mg/ml, added to cells 24 hours later, the cells which pricked membrane were 25.13.compared with the negative control group has notable effect(P<0.01).Conclusions: Identical saturation drug act to the cells, along with horary prolong, the proliferation inhibition ratio is upgrade. In the identical time, along with saturation drug increase, the proliferation inhibition ratio is also upgrade. Which presents time and saturation dependence. As well lower saturation drug acted to cells 48hours can repress cells'colony formation; acted to cells 24 hours can repress cells'invasion. |
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