The Influence Of Anemones Total Saponins Of Salivary Gland Adenoid Cystic Carcinoma Of High Pulmonary Metastasis Cancer Cell Apoptosis

Objective:Salivary adenoid cystic carcinoma is one of the most common malignant salivary gland tumors,the tumor has a strong invasion,invasive growth,early features of spreading and transfer along the nerve and blood vessel.Tumor metastasis through blood to the lungs.Can be taken orally,currently looking little side effect,and can be long-term used antitumor drugs has been one of the hot research of oral medical field.Salivary adenoid cystic carcinoma(salivary adenoid cystic carcinoma, SACC),is the most common malignant salivary gland tumors,the tumor has a strong invasion,invasive growth,early features of spreading and transfer along the nerve and blood vessel. Hematogenous metastasis to the lung cancer often and seriously endanger human health.Currently looking for oral,small side effects,long-term application of antitumor drugs has been one of the hot research of oral medical field.Anemone raddeana Regel(Anemone Raddeanin Regel) is the dried rhizome of Chinese Ranunculaceae plants,herbs pungent in flavor,heat, toxic,spleen after,has the efficacy of rheumatism,swelling and pain.Anemone raddeana Regel extract of total saponins of multiple anemone by hydrolysis and solvent extraction and. Study on the pharmacological activity of total saponin of Anemone found times have antitumor analgesic and sedative and anticonvulsant effects.In vitro experiments show the anemone raddeana Regel saponins of water soluble saponins on growth of rabbit brain c AMP-PDE has obvious inhibiting effect,up to a maximum of 61.3%. Another in vitro experiments showed that anemone raddeana Regel saponins can inhibit human cancer cells KB,HCT-8,MCF-7/WT and MCF-7/ADR,cell growth. Animal experiments results showed that anemone raddeana Regel saponins had obvious inhibition effect on S180 sarcoma,liver cancer H22 and Ehrlich ascites carcinoma EAC,its inhibitory rate was 68.1%,62.5%,69.3%.At present the effect of Anemone in adenoid cystic carcinoma has been reported less. This experiment adopts the method of hydrolysis and solvent extraction,Chinese medicine from anemone raddeana Regel extraction and isolation of total saponins. Based on the in vitro cultured Adenoid cystic carcinoma of high pulmonary metastatic(ACC-M),with different concentrations,different time of Anemone raddeana Regel saponins treatment,using the method of MTT,Giemsa staining,Annexin-V-FITC and PI double labeled cell apoptosis detection method of living cells,the effect of different concentration,different action time anemone raddeana Regel total saponin is there to induce the apoptosis of ACC-M cell line.Study on Chinese medicine metastasis of salivary gland adenoid cystic carcinoma cell effect on high pulmonary,provide a kind of effective method and way for prevention of metastasis of adenoid cystic carcinoma in the distance.Method: 1 material:ACC-M cells:Peking University Stomatological Hospital provides; Anemone raddeana Regel saponins:The Shijiazhuang lerentang drugstore(the origin of Heilongjiang).In the Hebei Medical University School of medicine by dissolution and solvent extraction purification. 2 methods: 2.1 Experimental preparation of drugs:Anemone raddeana Regel saponins prepared by DMSO dissolution of 20mg/m L storage solution.Before use and diluted with culture medium(DMSO<0.001%,had no effect on cell). 2.2 ACC-M cell culture:Cultured in 37℃,the 5%CO2 constant temperature box and saturated humidity conditions,10% fetal bovine serum(100U/m L) and penicillin,streptomycin(100g/m L) added to the culture medium of RPMI1640,24h~48h transmission of the 1 generation,With 0.04%EDTA and 0.25% trypsin(1:1) mixed,the mixed liquid used for digestion passage. 2.3 MTT colorimetric test:The reference cell growth curve,using the logarithmic growth phase were made of 2 × 107/L cell suspension,then inoculated into 96 pore plate,each hole 100 u L,24 h culture,in the experimental group added anemone raddeana Regel saponins 100 u L,make drug concentrations were 1ug/m L,25ug/m L,50ug/m L,75ug/m L,100ug/m L,and set the the control group and the zero hole,each with 5 holes.24 h,48h,72 h to culture,MTT solution 20 u L 5g/L per hole,continue to culture 4H to absorb all the supernatant of each hole,and then add the 200 u L DMSO solution in the hole,in the oscillator oscillation,the crystallization is fully dissolved,96 hole plate is arranged on the enzyme mark instrument measuring Kong Xi luminosity value and calculate the growth inhibition rate,different processing time for IC50 according to the mapping method(growth inhibitory rate as the drug concentration 50%). 2.4 Were observed by inverted phase contrast microscope:The ACC-M cells were cultivated in 50 m L flask,application of inverted microscope observation of ACC-M cells at different concentration and different culture time,the experimental group and the control group cells growth. 2.5 The results of optical microscope:ACC-M cells were seeded on coverslips 6 orifice,placed in the hole,the cell climbing piece success after adding blank or containing medium,after various incubation times,Giemsa staining,optical microscope. 2.6 Flow cytometry detection:Application of logarithmic growth phase cells,the cells were cultivated in 50 m L flask,time control group and the concentration of the control group,the Annexin-V-FITC and PI double labeling cells,apoptosis was detected by flow cytometry. 3 Statistical analysisUse the SPSS10.0 statistical software,growth inhibition rate and apoptosis rate is analyzed by mean deviation,T test,n=3, P<0.05Results:1 MTT colorimetric assay:Experiments show that the anemone raddeana Regel saponins(above 1ug/mg) has added significant inhibitory effect on ACC-M cells,Effects of 24,48,72 h after IC50 were about 46.3 ug/m L,56.59 ug/m L,44.60 ug/m L,The maximum inhibition rate of 90.3%(Figure 1).Statistical analysis results show,significant differences between the groups of different concentration of each time the absorbance value(P<0.05),Between each experimental group was treated for different time growth inhibition rate had significant difference(P<0.05).Anemone raddeana Regel saponins efficacy and its concentration and the reaction time has significant positive correlation.According to the time set of IC50 experimental design group of drug concentrations were 25ug/m L,50ug/m L,75ug/m L.2 Results under inverted microscope:The control group cultured cells adherent growth,is "slabstone" distribution.The cells were polygonal,plump cytoplasm,rapid growth,the nucleus can be seen near the center;The experimental group cells cultured adherent growth ability is abate,medium visible suspension cell shedding,adherent cells became round,cytoplasmic shrinkage,cells become small,growth slowed,the nucleus color deeper, increased cell refraction.3 The results of optical microscope:Control cells showed many nuclei,nucleoplasm is relatively high;The experimental group cells appeared cytoplasm condensed,nucleo cytoplasmic ratio decreased phenomenon,the nucleus decreased or even disappeared,chromatin arranged along the inner nuclear membrane.4 Flow cytometry results:The control group cells also appeared apoptosis,but the apoptosis rate is low.The experimental group cells were apoptosis,apoptosis rate increased with increasing with the prolongation of time effect of the drug concentration,drug concentration of 25ug/m L apoptosis rate increased significantly,the statistical analysis,there was significant difference between different concentration groups and each group(P<0.05).Conclusion:Anemone raddeana Regel saponins can inhibit the proliferation of ACC-M,can induce apoptosis in vitro,and was dose and time dependent.