| IntroductionToad venom is a major component derived from Chan Su,a kind of traditional Chinese medicine. It is generally called bufodienoHdes based on the same steroid nucleus. In previously study , toad venom may play a role of anti - tumor by inducing differentiation , improving apoptosis , inhibiting angiogenesis and altering gene expression correlated with tumor cell. An extract of partial purification was prepared from Chinese toad secrection ( Toad venom mixture , TVM ) , which could inhibit the cell proliferation of the human leukemic cell line HL60,K562,NB4, improve the ATRA action of inducing differentiation to the leukemia cell line NB4 and the primary culture cell of APL, induce apoptosis of HL60,K562,NB4,OVCAR3, downregulate Bcl - 2 protain expression and WT1 gene expression during human leukemic cell differentiation and apoptosis. But the mainly componentof anti - tumor and the anti - tumor mechanisms is still unclear.The purpose of this study was to determine the efficacy of the eth-anolic extract ( Toad venom mixture , TVM ) from Chinese toad secretion in treating kun - ming mice loaded H22 and S180 tumor cell.Materials and methods1. Materials and instruments1.1 Chinese toad secretion, parenteral solution of hua chan su (made in anhui jinchan corporation).1.2 Cell line: H22 cell line of mice hepatic tumor and S180 cell line of mice sarcom was provided by China Drug University.1. 3 Reagent; anhydrous alcohol, distilled water, Dimethyl Sulfoxide (DMSO) ,RPMI - 1640 culture medium, 10% fetal bovine serum, gentamycin, heparin sodium, sodium sulfide, H - E staining materials ect.1.4 Major Instruments; sigma 3k30 centrifuger, decompression stilling assembly,light microscope,depuration table,etc.1. 5 Animals: kun - ming mice was provided by China Medical University .2. Methods2. 1 Preparation of toad venom: We extract the secretion of toad sebaceous gland by special technique and dissolve them in double -stilling water, then centrifugate them. Put the depositor in anhydrous alcohol,then centrifugate them and decompress stilling,made them into shaf - powder at last, calculate the percentage of purification. 2. 2 Preparation of tumor cell; Putting the H22,S180 cell into RPMI 1640 supplemented with 10% fetal bovine serum after they were resuscitated. We took 0. 2ml cell suspension and injected them into intraperitoneal cavity. We drew ascites after 10 days , calculated and adjusted cell concentrition to 5 - 6 106 /ml with containing 0. 3 l/ml gentamycine and 3.2 J/ml heparin sodium.2.3 Preparation of mice loaded with tumor cell;2.3. 1 Preparation of mice loaded with ascites tumor cell: see 2. 2. Inoculating 0.2ml cell suspension(5 -6 106 /ml) into mice's intraperitoneal cavity.2.3.2 Preparation of mice loaded entity tumor: We cleared the villi with depilator containing sodium sulfide and then injected 0. 2ml cell suspension to subcutaneous tissue of right lower extremity.2.4 methods2.4.1 Subacute toxicity expriment of toad venom: All 80 mouse were divided 7 groups depending on their weight. TVM were administrated to mouse ranging from Img/kg to 160mg/kg for 9 days. The next day, we kill them and take out of bowel to embed in paraffin wax, make sections and stain with H - E staining.2.4.2 Experiment of mice loaded with H22 or S180 ascites tumor cell;We inoculated S180 tumor cells by intraperitoneal injections and the next day divided them into 7 groups. Experiment groups (3groups) were administrated TVM at 5mg/kg, 0.5mg/kg,0. Img/kg. Huachan-su groups were administrated 170mg/kg, 17mg/kg, 3. 4mg/kg used as positive groups and negitive group were given 0. 5% DMSO for 9 days. After stopping administration, we observed the life span and calculated extend rate of life span of load - tumor mouse. The mouse loaded H22 tumor cell were divided 3 groups and the dose is 5mg/kg of toad venom, 170mg/kg of huachansu and 0. 5% DMSO by sequence.2.4.3 Experiment of mice loaded with H22 or S180 entity tumor cell:We inoculated...
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