| Objective:1.To explored the effect of "Hua Tan Qu Yu Fang" on cellular morphology,cell proliferation,cell cycle,apoptosis,cell migration and invasion of Hep-2 and TU212 cells in laryngeal squamous cell carcinoma(LSCC).2.To explored the effect of "Hua Tan Qu Yu Fang " on signal transducer and activator of transcription 3(STAT3)and the downstream factors which are regulated by STAT3 in Hep-2 and TU212 cell lines of LSCC,so that to explain the mechanism of phenomena in objective 1.3.To established the transplanted tumor model of Hep-2 cell line in nude mice with phlegm coagulation and blood stasis syndrome,then conducted "Hua Tan Qu Yu Fang" to interfere the mice to verify the effect on the proliferation of Hep-2 cell line tumor with phlegm coagulation and blood stasis syndrome.4.By exploring the effect of "Hua Tan Qu Yu Fang " on STAT3 and downstream factors which are regulated by STAT3 in Hep-2 cell line tumor with phlegm coagulation and blood stasis syndrome,to verify the mechanism in vitro.Methods:1.CCK8 method was used to detect the IC50,optimal concentration and time on "Hua Tan Qu Yu Fang" treat Hep-2 and TU212 cells.2.The effects of "Hua Tan Qu Yu Fang " on the morphology of Hep-2 and TU212 cells were observed by inverted microscope.3.The effects of "Hua Tan Qu Yu Fang " on the proliferation of Hep-2 and TU212 cells were detected by CCK8 assay.4.PI flow cytometry was used to detect the effects of "Hua Tan Qu Yu Fang " on cell cycle of Hep-2 and TU212 cells.5.Fluorescence microscope was used to observe the changes of DNA replication in Hep-2 and TU212 cells after "Hua Tan Qu Yu Fang"intervention and Edu staining.6.Annexin-V-FITC flow cytometry was used to detect the effects of“Hua Tan Qu Yu Fang " on apoptosis of Hep-2 and TU212 cells.7.The apoptosis of Hep-2 and TU212 cells were observed by fluorescence microscope after treated with "Hua Tan Qu Yu Fang " and labeled with Hochest33342 staining.8.Inverted microscope was used to observe and record the changes of cell migration ability after "Hua Tan Qu Yu Fang" interfered with Hep-2 and TU212 cells.9.The invasiveness of Hep-2 and TU212 cells to Transwell cells which coated with matrix glue was observed and recorded by positive microscope after intervention of "Hua Tan Qu Yu Fang".10.The different concentrations of "Hua Tan Qu Yu Fang " were used to intervene Hep-2 and TU212 cells of LSCC for 48 h,respectively,than STAT3,p-STAT3,CyclinDl,CyclinBl,P27,Bcl-2,Bax,Caspase-3,E-cadherin and MMP-9 proteins were detected by Western Blot.11.Conducted "combination of disease and syndrome" method which was establishment of animal model by building up Hep-2 cell transplanted tumor nude mice with phlegm coagulation and blood stasis syndrome.The nude mice model of phlegm coagulation and blood stasis syndrome were established by feeding with high fat and high cholesterol diet+0.1%epinephrine hydrochloride subcutaneous injection+ice water swimming stimulation,and then Hep-2 cells were injected subcutaneously.12.The Hep-2 cell transplanted tumor nude mice with phlegm coagulation and blood stasis syndrome were divided into control group(Con),“Hua Tan Qu Yu Fang" low dose group(Low),“Hua Tan Qu Yu Fang”middle dose group(Mid),"Hua Tan Qu Yu Fang" high dose group(Hi)and cisplatin group(Cis),randomly,6 mice in each group.Among them,mice of Con group were given normal saline 0.2mL intragastrically once a day for 3 weeks.Low,Mid and Hi groups ’ mice were given "Hua Tan Qu Yu Fang”intragastrically once a day for 3 weeks,and cisplatin was injected intraperitoneally once a week for 3 weeks in Cis group’ s mice.During the administration,the general state and signs,drinking water,food intake and body weight of nude mice,and the changes of tumor volume of nude mice in each groups were measured.13.When drug intervention finished,the nude mice in each group were euthanized and transplanted tumors were extracted.The mRNA expressions of STAT3,Bcl-2,Bax,P27,CyclinDl and CyclinBl in tumors of each group were detected by RT-PCR and the protein expressions of STAT3,P27,CyclinDl and CyclinBl were detected by Western Blot.Results:1.After the intervention of "Hua Tan Qu Yu Fang" for 0,0.8,1.6 and 3.2mg/mL in Hep-2 and TU212 cells for 48h,with the increase of drug concentration,the number of the two cell lines decreased gradually,the density of the two cells was sparse,and the proliferation of the two cells slowed down.The cells crumpled and became smaller.2.After "Hua Tan Qu Yu Fang" interfered with Hep-2 and TU212 cells for 12h,24h and 48h respectively,the proliferation of the two cell lines was inhibited(P<0.05),and showed a concentration-time-dependent effect.3.After intervention of "Hua Tan Qu Yu Fang" on Hep-2 and TU212 cells for 48h,the proportion of G0/G1 phase cells in the two cell lines increased(P<0.05),S phase cells decreased(P<0.05)and G2/M phase cells decreased(P<0.05),the cell cycle of both cell lines was inhibited in G0/G1 phase.4.After intervention of "Hua Tan Qu Yu Fang" on Hep-2 and TU212 cells for 48 hours,the proportion of DNA replication cells in these two cell lines decreased(P<0.05).5.After intervention of“Hua Tan Qu Yu Fang”on Hep-2 and TU212 cells for 48h,the proportion of apoptotic cells increased with the increase of drug concentration(P<0.05).6.After intervention of“Hua Tan Qu Yu Fang”on Hep-2 and TU212 cells for 48h,the nuclei of the two cell lines was concentrated and stained bright blue,the nuclei was lobulated and segmented or apoptotic bodies.The proportion of these cell increases with the concentration of intervention drugs.7.Hep-2 and TU212 cells ability of cell migration was inhibited after "Hua Tan Qu Yu Fang" interfered with(P<0.05).8.The number of cells which passing through the matrix glue coating Transwell chamber of Hep-2 and TU212 cells were decreased with the intervention of“Hua Tan Qu Yu Fang”for 48 hours,respectively.9.The expression of STAT3,p-STAT3,CyclinDl,CyclinBl,Bcl-2 and MMP-9 proteins were down-regulated in Hep-2 and TU212 cells whiche treated with“Hua Tan Qu Yu Fang”,respectively(P<0.05).Howerver,the expression of P27,Bax,Caspase-3 and E-cadherin proteins were up-regulated by "Hua Tan Qu Yu Fang"(P<0.05).10.Before interention,there were no significant difference in body weight,drinking water and food intake between the model control group mice and the model group mice(P>0.05);After modeling,there were also no significant difference in body weight,drinking water and food intake between the two groups’ mice(P>0.05).But blood lipid function of nude mice in the two groups were vary(P<0.05).The total cholesterol in the model group’ s mice were higher than that in the model control group(P<0.05).High density lipoprotein(HDL)in the model control group’ s mice were higher than that in the model group(P<0.05),but there was no significant difference in coagulation function between the two groups’ mice(P>0.05).11.After drug intervention,the growth rate of Hep-2 transplanted tumor in nude mice with phlegm coagulation and blood stasis syndrome was inhibited by,and compared with Con group,Hi group and Cis group had significant inhibitory effect(P<0.05).12.Compared with Con group,the mRNA expression of STAT3,Bcl-2,CyclinDl and Bax in Low group,Mid group,Hi group and Cis group,were down-regulated(P<0.05),while the P27 was up-regulated(P<0.05),but the CyclinB1 was not significantly changed(P>0.05).13.Compared with Con group,the protein expression of STAT3,CyclinDl and CyclinBl in Low group,Mid group,Hi group and Cis group decreased(P<0.05),while the expression of P27 protein in Low group,Mid group and Hi group increased(P<0.05).Conclusion:1."Hua Tan Qu Yu Fang" inhibits the proliferation and DNA replication of Hep-2 and TU212 cells in a concentration-dependent manner.One of the mechanisms may be through inhibiting STAT3 signaling pathway.2."Hua Tan Qu Yu Fang" inhibits the cell cycle of Hep-2 and TU212 in G0/G1 phase in a concentration-dependent manner.One of the possible mechanisms was down-regulation expression of CyclinDl and CyclinBl of downstream factors of STAT3 signaling pathway and up-regulation of P27 expression.3.“Hua Tan Qu Yu Fang" may induces apoptosis by down-regulating the expression of Bcl-2,a downstream factor of STAT3 signaling pathway,but up-regulating the expression of Bax and Caspase-3 in Hep-2 and TU212 cells of LSCC in a concentration-dependent manner.4."Hua Tan Qu Yu Fang" can inhibits the migration and invasion of Hep-2 and TU212 cells in a concentration-dependent manner.One of the mechanisms may be to down-regulate the expression of MMP-9 while up-regulate E-cadherin proteins in the downstream factors of STAT3 signaling pathway.5.Experiments results confirmed that "Hua Tan Qu Yu Fang" inhibited the proliferation of Hep-2 by down-regulating the expression of STAT3 and its downstream factors CyclinDl and CyclinBl,and up-regulating the expression of P27. |
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