Effects Of Sodium Phenylbutrate On Invasive Ability Of Human Thyroid Follicular Carcinoma Cell

Objective:To investigated the effect and mechanism of infiltration and metastasis of sodium phenylbutyrate(NaPB) on CGTHW-1 cell line through studing invasion ability and the secretion and transcription of MMP-9 and TIMP-1,Providing a theory evidence for the drug treatment of thyroid carcinoma.Methods:Phase 1: CGTHW-1 cell was cultivated with 1640 medium containing 10% fetal calf serum.when the bottom was covered with cells, digested and separated the cells with Trypsin,centrifugated and then inoculated the cells in two or more bottles,Continued to cultivate and subculture.Phase 2: CGTHW-1 cell which was in exponential phase of growth was inoculated at 96-well plates,every group was treated with a certain concentration of NaPB,such as 1mmol/L,2mmol/L,3mmol/L,4mmol/L,5mmol/L,and so on.Then cultivated for 24 h,48 h and 72 h,discarded the supernatant, added 200μl DMSO in each hole, Shaken.determined optical density(OD) value by Universal Microplate Spectrophotometer., calculated the Inhibition rate of CGTHW-1 cell from NaPB,then determined the best action time and drug concentration of CGTHW-1 cell.Phase 3: CGTHW-1 cell was divided into two groups, control group without any interfering factors and experimental group treated with 4mmol/L NaPB for 72 h. Put matrigel on the polycarbonate membrane of transwell chamber,CGTHW-1 cell was in epicoele and the supernate of 3T3 cell was under the chamber.Cultivated the cell for 20-24 h. Stained them with hematoxylin and eosin When the cells went straight through matrigel and appeard under the chamber. Counted the number of cells which had gone straight through matrigel,the ability of invasiveness was defined as the average cell number of 5 field of vision.Phase 4: CGTHW-1 cell was treated with different density of NaPB for 72 h(control group,4 mmol/L NaPB group,6 mmol/L NaPB group).Conducted immunohistochemical staining,make photographs under the light microscope,analyzed the OD of cells, investigated secretion of MMP-9 and TIMP-1 through Statistical analysis of average OD.Phase 5: Choose 5 sample,every sample was divided into 2 groups,control group without any interfering factors and experimental group treated with 4mmol/l NaPB for 72 h. Extracted total RNA,conducted reverse transcription reaction. The amplification product was used for electrophoresis in 1.0% agarose gel. Make photographs and analyzed their OD.Then investigated the transcription of MMP-9 and TIMP-1 through Statistical analysis of average OD.Results:(1)Sodium phenylbutyrate depress CGTHW-1 cells invade through matrigel.(2) Protein expression of MMP-9 and TIMP-1 were down regulated under sodium phenylbutyrate(p<0.05).(3)mRNA expression of MMP-9 and TIMP-1 were down regulated under sodium phenylbutyrate(p<0.05)Conclusion:Sodium phenylbutyrate depress CGTHW-1 cells invasion by inducing the down-regulation of MMP-9 and TIMP-1 expression.