Delisheng Injection Collaborate With 5-FU Induce The Apoptosis Of HCC Cell Line HepG₂ In Vitro

Objective:From cells apoptosis morphologic observation to the research of molecular level,Elaborate Chinese medicine Delisheng(DLS) to coordinate the chemotherapy medicine 5-fluorine(5-FU) on apoptosis of HepG₂,and initial discusses the mechanisms of apoptosis induction.Thus treats liver cancer for the clinical association with DLS to provide the theory basis and to further study the cooperation of Chinese medicine and Western medicine treatment tumor to provide the theory instruction.Methods:1.Cell culture and apoptosis model building:Transfers generation to raise the HepG₂ cell,according to the request and the packet of experiment process HepG₂ cell,establishes apoptosis model.2.MTT experiment:observes the proliferation of DLS coordinate 5-FU to HepG₂ cell,which is evaluated by MTT assay.And see both union medication effect.3.Cytomorphology observation:use the inverted microscope observant HepG₂ cells morphologic changes without dyeing;Uses the fluorescence microscope observation the HepG₂ cell which dyes by Hoechst 33258.4.Flow Cytometry(FCM):After experiment packet processing HepG₂ cell 12h, double dyes FCM through AnnexinV/PI to examine the HepG₂ mitotic cycle distribution,and unifies the morphology change.5.TdT 2-ediated dUTP nicked labeling technique(TUNEL):Observes using the TUNEL under the fluorescence microscope perishes cell's occurrence,to detect apoptosis occurrence,take photographs and calculates Apoptosis index(AI).Result:1.MTT experiment:The MTT examination indicated that DLS promotes 5-FU to the HepG₂ cell's growth and the multiplication inhibitory action, this function assumes the obvious effectiveness and the quantity effect relations;Independent DLS application and union 5-FU IC₅₀ respectively are 0.82μg/mL and 0.32μg/mL,unites the medication can to reduce IC₅₀ obviously;Merge index CI₅₀ is 0.73,the efficiency multiple is 2.56, the union medication has the synergism.2.Cytomorphology observation:Under the inverted microscope observes normal group cell to paste the wall to grow,crowded,big nucleus,visible faintly many nucleolus's,after the medication affects 24h,the cell volume changes small,changes circle,separates with the neighbor cell,cytoblastema concentration;After Hoechst dyes fluorescence observation:The normal group cell nucleus dyes homogeneous,the structure complete uniformly,assumes the normal blue color,the medication group displays apoptosis cell characteristic change,the cell nucleus presents compact dyes or the fragments thickly compact dyes thickly,dyes some blanches,presents the karyopyknosis and nucleus decomposition phenomena.In tests in the density scope,two observation methods demonstrated that increases this kind of phenomenon along with the medicine density and the time to be more obvious, moreover under the homogeneous condition,the union group phenomenon is more prominent.3.Flow Cytometry(FCM):According to experiment packet processing HepG₂ cell 12h,the number of apoptosis cell of normal group account for 3.40%,and hasn't appeared the apoptosis peak,the medicine treatment group has the peak.Double dyes FCM through Annexing/PI to observe each medication group Flows type chart,Some apoptosis peakappearance ahead of G₁time,and increases along with the density assumes increases the tendency;the G₂/M time cell's percentage along with the density to increase gradually,the union group hinders in the G₂/M time to be more obvious.According to each group of cell histogram analysis,the 5-FU group,the DLS group,unite A group,unite B group,unite C group of apoptosis rate are in turn 21.30%,18.89%,22.01%,24.6%,39.2%,induces apoptisis function assume the quantity effect relations,The union group apoptisis rate increase obviously.4.TdT 2-ediated dUTP nickend labeling technique(TUNEL):The TUNEL masculine signal performance for olivine color strong fluorescence,located at the nucleus,assumes the circumduction,the small circular or granμlated,moreover many models "soak" the cell which obviously forms,tn density scope of test, this kind of olivine color fluorescence intensity change assumes the quantity effect relations.Other each group is quite even with the normal group has the remarkable difference(P＜0.01),Unites A group with to unite C group to be quite even with the 5-FU group has the remarkable difference(P＜0.01),Unites C group to have statistics significance with the 5-FU group comparison(P＜0.05).Conclusion:1.DLS not only itself may induce the HepG₂ cell to apoptisis,moreover can promote 5-FU to induce the apoptisis of HepG₂ cell;Use some method can exanimate the HepG₂ cell apoptosis occurrence,qualitatively and quotably.2.DLS,5-FU Shan Yonghe unites with suppresses to the person liver cancer HepG₂ cell multiplies and induces perishes the functionto assume the good time dependence weakly and the dosage dependence.3.The small dosage 5-FU merges low toxic DLS to be able to achieve the inhibition effect of toxic 5-FU which need high dose,thus manifests merit of the union medication fully:The small dosage,the low toxicity,highty effective.4.Through cytotoxin function damage the DNA of cancer cell,start programmed cell death,which is one of inhibitory action mechanisms to the HCC HepG₂ cell; Hinders the cell multiplication cycle,which is one of the mechanisms of traditional Chinese medicine regulation tumor cell.