The Research Of Saffower Polysaccharides On Human Tongue Squamous Carcinoma HN-6 Call Intervention Mechanism

Objective:Safflower polysaccharide(safflowerpolysaccharide,SPS)in the body,outside intervention in human tongue squamous carcinoma(tongue squamous cell carcinoma,TSCC)HN-6 cell proliferation and induce apoptosis and related gene and protein expression.For exploring the low poison,the highly effective intervention for the treatment of human tongue squamous carcinoma of TCM drugs to provide theoretical and experimental basis.Method:In vitro studies:1.The use of CCK 8 colorimetry to detect different concentrations of SPS and cisplatin in the different time intervention effect on HN-6 cell growth.2.With inverted microscope SPS(observation of cell morphology change);And using AO/EB double color fluorescent dye safflower polysaccharides and cisplatin on human tongue squamous carcinoma HN-6 cells apoptosis.3 Flow cytometry(FCM),application of SPS intervention posterity tongue squamous carcinoma HN-6 in the cell cycle when the cell percentage changes and the impact on the cell apoptosis4 Western blot test after SPS intervention caspase-3、COX-2,Bcl-2,Bax protein expression5 Real-time quantitative PCT SPS(QRT-PCR)method for caspase-3、COX-2,Bcl-2 及 Bax gene expression level.in vivo test:1 the human tongue squamous carcinoma HN-6 cells build nude mice transplantation tumor model.After a tumor were randomly divided into model set cisplatin group(5 mg/kg)SPS high concentration group(80 mg/kg),SPS concentration group(40 mg/kg),SPS,low concentration group(20 mg/kg)group,a total of 5 groups,each group of four.Experimental group in nearly every day back subcutaneous tumor place local tissue injected dosing,and the dosing metering is 0.8 ml/d,1/d,continuous dosing 20 d,diet,activities and mental health of every observation nude mice.Put to death at the end of the treatment of mice,stripping acquisition and calculate the inhibitory rate of tumor weighing.2 application QRT-PCR technique to detect COX-2,Bcl-2 and BaxCleaved caspase-3mRNAusing semi-quantitative analysis was carried out on the test results,thereby understand SPS inhibition of tongue squamous cancer cells transplantation tumor.3 Westernblot method is used to detect transplanted tumor of Cleaved caspase 3,cox-2,the Bcl-2 and Bax protein expression,andsemi-quantitative analysis was carried out on the test results,further studies SPS,tongue cancer cells transplantation tumor proliferation and promote apoptosis mechanism.Resultsvitro experiment:1 CCK 8 colorimetric method shows red polysaccharides and cisplatin on human tongue squamous carcinoma HN-6 cells proliferation have different degrees of inhibition,compared with the blank control group was statistically significant difference(P<0.05),and Safflower polysaccharide enhanced inhibition increases over time,and within a certain concentration,enhanced with the increase of concentration,and presents the concentration-response relationship,when in 0.64 mg/ml,inhibitory effect is most obvious.2 the application of inverted microscope to observe safflower polysaccharide and cisplatin after 24 h shows:HN-6 cell proliferation slowing,adherent cells decreased,intercellular space increased,and part of the cell,it become smaller,nuclear chromatin density,some cells in the cell membrane and cytoplasm vacuolated particles increased obviously,part of the floating,part of the cell disruption in the culture rupture,and apoptotic body formation.With the increase of concentration of SPS,the change is more obvious.3 using AO/EB double color fluorescent dye detection,results safflower polysaccharide and cisplatin induced human tongue squamous carcinoma HN-the role of cell apoptosis;By the flow cytometry instrument detection,HN-6 cells by 0.64 mg/ml SPS effect after 24 h,the late early apoptosis rate,cell necrosis or apoptosis rate increased significantly,GO/G1 phase cell proportion increased significantly,and the proportion of S stage cells down.4 Western blot detect:SPS role HN-6 activation of the Cleaved caspase 3 apoptosis expression mechanism,the Bcl-2 and reduced the volume of cox-2 protein expression,on the contrary Cleaved caspase 3,increased Bax protein expression,Compared with the blank control group difference is statistically significant.5.QRT-PCR exist cox-2 experiment found that the experimental group and control group,the Bcl-2 and Bax mRNA expression,comparing the control group,the Bcl-2 and reduced the volume of cox-2 gene expression,Bax gene expression increased compared with control group(P<0.05.Vivo experiment::1 compared with the blank control group,safflower polysaccharide group and cisplatin control tumor growth is slow,as the growth of the SPS medication time,tumor inhibition effect is more obvious;By the end of the treatment,each group nude mice transplanted tumors present product,the quality of tumor,there were significant differences(P<0.05).2 the quality of tumor,and tumors had product inhibition rate of 45.65—51.72%;The whole treatment process,at least no obvious adverse reaction,at the end of the treatment,each group of nude mouse body smooth quality compared with the treatment of precursor quality increase,the ratio is greater than 0.8,no obvious toxic effects.3 optical microscope display:cisplatin controls within the tumor tissue necrosis collapse area significantly greater than the control group,alien is smaller than the control group,in the form of cell apoptosis phenomenon such as visible nuclear pyknosis.4 QRT-PCR technique to detect transplanted tumor of cox-2,the Bcl-2 gene expression,Bax and Cleaved caspase 3 gene expression to rise,with statistical difference(P<0.05);5 QRT-PCR technique to detect cox-2 of transplanted tumor,cut the Bcl-2 gene expression,Bax and Cleaved caspase 3 mRNA expression level,with statistical difference(P<0.05).Conclusion:1 Safflower polysaccharide and cisplatin can inhibit human tongue squamous carcinoma HN-6 cells and nude mouse transplantation tumor proliferation,And within a certain concentration,with the increase of the concentration effect,present a certain effect.2 Safflower polysaccharide can promote human tongue squamous carcinoma HN-6 cell nude mouse transplantation tumor apoptosis.3 Safflower polysaccharide can control human tongue squamous carcinoma HN-6 cell cycle,main show is cell cycle arrest in GO/G1 phase.4 safflower polysaccharide can cut the tongue squamous carcinoma HN-6 cell and nude mouse transplantation tumor cox-2,the Bcl-2 protein and mRNA expression,raise Bax Cleavedcaspase-3 mRNA and protein expression.