Dissertation
Dissertation > Medicine, health > Oncology > Oral cavity, maxillofacial tumors

Effect of NF-κ Bp65siRNA on the Proliferation, Apoptosis and Drug Resistance of Oral Squamous Cell Carcinoma Cell Line Tca8113

Author WangYun
Tutor GongLi
School Luzhou Medical College
Course Pathology and Pathophysiology
Keywords Oral squamous cell carcinoma NF-κBp65 siRNA Cell proliferation Cell apoptosis Drug resistance
CLC R739.8
Type Master's thesis
Year 2013
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Abstract:Objective:Using siRNA to interferent the NF-κBp65gene within oral squamous cell carcinoma cell line Tca8113, and combining with5-Fu,then observing the effects of NF-KBp65siRNA on Tca8113cells proliferation, apoptosis and the impact of drug resistance, to provide new ideas for the treatment of oral squamous cell carcinoma. Methods:The Tca8113cells were cultured in vitro and divided into three groups when they were in exponential growth phase:①blank control group;②egative control group;③experimental group.1. NF-κBp65siRNA was transiently transfected into Tca8113cells, observing the cells in each group with fluorescent microscope, and canculating the rate of transfection;2. The expression level of NF-κBp65mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR);3. The expression level of NF-κBp65protein was determined by enzyme-linked immuno sorbent assay (ELISA) after transfection;4. The proliferation of Tca8113cells was detected by methyl thiazolyl tetrazolium (MTT) after transfection;5. The apoptosis of the Tca8113cells was detected by Annexin V-FTIC/PI;6. Tca8113cells were divided into transfection group and non-transfection group after transfection, then combined with different concentration of5-Fu (Omg/L,16.35mg/L,32.7mg/L,327mg/L,3270mg/L,6540mg/L), the sensitivity of Tca8113cells after cultured72hours to5-Fu was detected by MTT. Results:1. A fluorescein-labeled NF-κBp65 siRNA was used tomonitor the efficiency in Tca8113cells, demonstrating nearly61.1%transfection efficiency24hours post transfection.2. The test results of semi-quantitative RT-PCR showed that,48hours post transfection, the relative quantitative expression of NF-KBp65mRNA was0.539±0.036in cells of blank control group,0.514±0.031in cells of negative control group,0.313±0.031in cells of experiment group, the relative quantitative expression of NF-κBp65mRNA in cells of experiment group decreased significantly compared with the other tow groups, a statistically significant difference (P<0.05).3. The test results of ELISA showed that,48hours post transfection, the relative quantitative expression of NF-κBp65protein was1.165±0.074in cells of blank control group,1.182±0.049in cells of negative control group,1.002±0.055in cells of experiment group, the relative quantitative expression of NF-KBp65protein in cells of experiment group decreased significantly compared with the other tow groups, the difference was statistically significant (P<0.05).4. The MTT results displayed that, the absorbance values of cells in experiment group were lower during each timing period compared with the other tow groups (P<0.05), the proliferation of Tac8113cells was inhibited obviously, and the inhibitory effect is highest at72hours post transfection, the rate of cell inhibition was28.1%.5. The test results of Annexin V-FITC/PI showed that,72hours post transfection, the apoptosis rata was (8.50±0.92)%in blank control group,(8.39±1.03)%in negative control group,(24.58±2.31)%in experiment group, the apoptosis rata of experiment group was higher obviously than other tow groups, a statistically significant difference (P<0.05), and the early apoptosis rate was greater than late apoptosis rate.6. Tac8113cells were combined with different concentration of5-Fu after transfection, the results of MTT showed that, the proliferative activity of cells in transfection group and non-transfection group had a downward trend along with increasing concentration of5-Fu72hours post transfection, and the proliferative activity was lower in transfection group than in non-transfection group at the same concentration, a statistically significant difference (P<0.05),it meaned that the sensitivity of cells to5-Fu in ransfection group increased significantly compared with non-transfection group. Conclusion:1. NF-κBp65siRNA could effectively transfect the oral squamous cell carcinoma Tca8113cells.2. The expression level of NF-KBp65mRNA and protein within the oral squamous cell carcinoma Tca8113cells decreased obviously after transfection, this displayed that the expression of NF-KBp65gene in oral squamous cell carcinoma Tca8113cells was silenced by NF-KBp65siRNA.3. Silencing the NF-κBp65gene could inhabit the proliferation of oral squamous cell carcinoma Tca8113cells, and induce oral squamous cell carcinoma Tca8113cells apoptosis.4. After the NF-KBp65gene was silenced, the sensitivity of oral squamous cell carcinoma Tca8113cells to5-Fu was significantly improved.6. NF-κB may be emphasized as a therapeutic target for gene theary of oral squamous cell carcinomas.

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