| Oral squamous cell carcinoma (OSCC), that has a predilection for tongue, is the most common malignant tumor occurred in the oral maxillo-facial region, counting for almost80%of the malignant tumors occurred in this region according to the reports. Unfortunately, all the current treatments for OSCC are unable to remove tumor cells completely. As the establishment of cancer stem cell (CSC) theory, the treatment for tumor has entered a new era and the basic principles of cancer therapy have been changed. Targeting cancer stem cells in order to completely block tumor recurrence and metastasis has turned into a hot spot nowadays. Accumulating evidence suggests that special gene therapy has good application prospect for malignant tumor treatment in the future. RNA interference technique is one of the outstanding achievements in this field. It has been used to block tumor related genes in order to explore the pathogenesis of tumor and synthesize siRNA related drugs which would be applied to the gene therapy for tumor.p75neurotrophic factor receptor (p75NTR), molecular weight75KDa, is one of the cell surface receptors. The expression of P75NTR in many tumor types has been discovered and confirmed. The expression and invasional characteristics of p75NTR positive cells is closely related with the prognosis of tumor. p75NTR has been regarded as a diagnostic indicator by many researchers. Through previous studies, we found that p75NTR was expressed by tongue OSCC cells and confirmed that p75NTR positive cells possessed the same proliferation and differentiation characteristics as that of the stem cells. It is necessary to explore the role of p75NTR in proliferation or apoptosis of the tongue OSCC cells in further study. The signal pathway and the function of p75NTR in the proliferation and apoptosis of tongue OSCC cells is important for curing tongue OSCC and the development of CSC-targeted anticancer drugs.ObjectiveFor specific genes to treat tongue squamous carcinoma with genes and looking for providing theoretic basis of anticancer drug, We try to explore the pathway of p75NTR mediated oral tongue squamous cancer cells apoptos is at the molecular level.Method1. The culture of tongue squamous cancer cells and the sorting of cells pos itive for P75NTR Tca8113cell lineage was cultured in RPMI-1640medium, at37℃and5%CO2, saturated humidity incubator. The sorting of p75NTR positive cells was finished by flow cytometry. The morphology and activity of cells were ob served under inverted phase contrast microscope.2. Down regulation of p75NTR protein expression in tongue squamous canc er cells by siRNA interference technologysiRNA was designed according to human being p75NTR gene (CD271Gen Bank Accession Number:NM-002507) sequence and three siRNA sites were ch oosen. P75NTR positive cells were divided into blank group, negative group an d experimental group according to.with or without siRNA transfection. Transfe ction efficiency was detected by flow cytometry after48h. Cell apoptosis chan ges of each group were examined by annexin V/PI double dye and PCR was used to detect p75NTRmRNA interference effect, and western blot was used to detect p75NTR protein expression.3. The changes of Bcl-2expression after transfection were detected by western blot. 4. The distribution of NF-κB in cells were observed by cell immunofluorescence labeling method.As required for transfection of plasmid itself green fluorescent labels, theref ore the NF-κB of protein with PE (red fluorescence) marked the second antis ubstance. DAPi labeled the nuclei are blue. Observing red particles distribution characteristics inside the cell under inverted fiber mirrorResult1. p75NTR and its mRNA expression was down-regulated by the transfection of siRNA. The cell apoptosis rate of the experimental group was statistically increased compared with that of the control group.2. Cell immunofluorescence labeling showed that NF-κB located mainly within the nucleus in the control group, while more NF-κB located in cell cytoplasm in the interference group.3. Western blot results showed that the Bcl-2protein expression of interference group was increased. Conclusion1. p75NTR protein expression was down-regulated successfully in tongue sq uamous cancer cells using siRNA technology, p75NTR has a "positive" bi ological effects in tongue squamous cancer cells2. Molecular mechanism of p75NTR mediating tongue squamous cancer cells apoptosis has some relation with activation of NF-κB.3. Western blot detect the BcL-2expression increased with tongue squamou scancer cell apoptosis increases. The BcL-2protein did not involve in cells apoptosis however BcL-2protected cancer cells... |
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