| The mechanism for malignant transformation of epithelial cells into squamous cell carcinoma has been focused in recent decades, among which the proliferation and apoptosis theory of epithelial cells is accepted extensively, and confirmed by series of investigations. It has been proved that malignant transformation of epithelial cells is induced by the microenvironment. Whether there are one or more key points to affect the canceration process of epithelium is investigated in this study. To find the critical site which determined the malignant transformation of epithelia by experimental investigations will benefit the prevention and interruption of canceration. The Potassium Ion Channel of Kv3.4which was very critical in the micro-environment investigated in this study and molecular biotechnical technique is taken to provide theoretical and clinical information for subsequent research and clinical therapy. Objective:1. To investigate the relationship between the content of Kv3.4protein and Kv3.4mRNA in cellular ion channel.2. To investigate the relationship between ion channel Kv3.4and the development of oral squamous cell carcinoma. Methods:1. To build the oral mucosa cell canceration model with60Sprague-Dawley (SD) rats:feed the50SD rats with4NQO solution (40ppm) to induce consecutive canceration as experiment group;10SD rats as control group.2. Identification of the canceration model:fix the obtained mucosa tissue, H&E staining and continuously identify the canceration process.3. Determination of the Kv3.4protein:The SP Immunohistochemistry (IHC) method was taken for serial sections to determine the content of Kv3.4protein with an antibody concentration of1:400. The differences in expression of Kv3.4were analyzed by using the Kruskal-Wallis Test and Mann-Whitney Test (α=0.01).4. Reverse transcription polymerase chain reaction (RT-PCR) was involved to determine the changes of mRNA in the frozen tissue. Bio-Rad image system was used for data collection, and SPSS Ver.19.0was used for statistical analysis. Results:1.16cases of normal buccal mucosa,10cases of mild dysplasia,12cases of moderate dysplasia,11cases of severe dysplasia and9cases of buccal carcinoma were detected with H&E staining detection. Symptoms of epithelial dysplasia, like loose density and polarity loss of epithelium, dropwise epithelial rete pegs, increased mitotic figures, increased hyperchormatic nuclei and hyperkeratosis of spinous cells conformed histopathology characteristics.2. Kv3.4protein determination revealed Kv3.4expression in cytoplasm and plasmalemma, occasional expression in nuclei, positive expression in gland and blood vascular endothelium, but no expression in intercellular. Content of Kv3.4protein increased with epithelium dysplasia, and was detected positive in buccal carcinoma. Significant statistical difference (P<0.01) was detected for Kv3.4expression in normal buccal mucosa, epithelium dysplasia and buccal carcinoma, but not in mild dysplasia, moderate or severe dysplasia(P>0.01). An increasing trend of Kv3.4protein from normal buccal mucosa to epithelial dysplasia and buccal carcinoma was detected.3. RT-PCR determination to mRNA revealed that:Significant statistical difference was detected of Kv3.4mRNA among normal buccal mucosa, epithelium dysplasia and buccal carcinoma (P<0.05). The same as the Kv3.4protein, there was a gradually increasing trend from normal buccal mucosa to epithelial dysplasia and buccal carcinoma. No significant difference was detected in epithelium dysplasia (T test, P>0.05). Conclusion:1. Content of Kv3.4protein increased with the severity of epithelium dysplasia;2. Content of Kv3.4protein increased at different levels in the process of epithelium dysplasia to buccal carcinoma;3. Kv3.4mRNA maintained the same changing pattern with protein. |
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