| Objective:Oral cancer accounts for 3%-5% in systemic malignant tumor, ranking the second in head and neck malignant cancers. About 90% of oral cancer is squamous cell carcinoma, with 5-year survival rate of approximately 50%. It is generally believed that oral squamous cell carcinoma (OSCC) is mainly developed from the oral precancerous lesions. Early discovery, early diagnosis and early treatment are essential to increase the survival rate and life quality of the patients with OSCC nowadays. In recent years, with the development of medical science and technology, the tumor markers, as a new means of cancer diagnosis, plays a unique role for early diagnosis, becoming a new area of concern in oncology. At present, it has not found the specific, sensitive indicators yet. Selecting some highly specific tumor markers to detect a certain kind of cancer diagnosis will be better than a single indicator. Cytokeratin (CK) 19 is a kind of acidic polypeptide, which is expressed in the epithelial layer of epithelial cells, and in basal cells of the stratified squamous epithelium. Many domestic and foreign researchers have detected CK19 in various cancer tissues. The abnormal expression of CK19 can be used as a marker for auxiliary diagnosis and differential diagnosis of carcinoma. In oral carcinogenesis, CK19 expression increased with the exacerbation, it can be used as auxiliary diagnostic indicators of OSCC differentiation. The higher the degree of malignancy of oral squamous cell carcinoma, the stronger the CK19 expression. Tumor is an uncontrolled proliferation and differentiation disorder. Block of any step in the developing process may curb the tumor's development. The previous researches had proved the universal defects of gap junctional intercellular communication (GJIC) in the tumors and transformed cells. In most tumors, the function of GJIC decreases or loses, with aberrant location of Cx43 protein, even loss of expression. Restoring GJIC can inhibit tumor, making GJIC a potential breakthrough to prevent and treat tumor. The scholars believe that in the process of tumor formation, the functional defect or loss of GJIC makes cancerous cells lack of the growth regulation of the neighboring normal cells, leading to unlimited growth and eventually developing into a tumor. Connexin43 (Cx43) is the most widely distributed connexin, drawing wide attention and undergoing in-depth researches. GJIC defects and tumor development are closely related, but there is little information about the relationship between GJIC defects and oral mucosa. The experiment plans to use 4-nitroquinoline-l-oxide (4NQO) to induce oral carcinogenesis of SD rats, and to obtain the tongue organization from each stage, such as'the normal oral mucosa, precancerous lesions and oral squamous cell carcinoma. In this study, immunohistochemical SP method was used to detect the expression of CK19 and Cx43 in various stages of oral carcinogenesis. The author of this thesis also analyzed the relationship between CK19 and Cx43 in oral carcinogenesis to decide whether there is a connection between CK19 and Cx43 in the process of oral cancer. Methods:Fifty-eight male Sprague-Dawley (SD) rats weighing about 200±10g,7 weeks old, were used as subjects. The rats and the food were obtained from the Animal Experimental Center of Luzhou Medical College. They were housed, three or four rats in one plastic cage, with hardwood chips for bedding in an experimental room with temperature controlled at 23±2℃, the humidity 55±5%, and lightening 12-h light/dark cycle. Experiment group (48 rats) was given drinking water containing 40ppm 4-NQO and basal diet until being sacrificed, and the other 10 rats served as the control group without 4-NQO. The forty-eight experimental rats were respectively killed at the end of the 9th,12th,18th and 22nd weeks after the commencement of the experiment. The left ten rats, which were feed without 4-NQO, were killed at the end of the experiment. Extract the oral mucosa from the 58 rats respectively. Fixed with 4% neutral formaldehyde, embedded in paraffin, and 4μm thick serial sections were cut. Two experienced pathologists blinded method by HE staining for pathological diagnosis. With rabbit-anti-rat CK19 (M259) and rabbit-anti-rat Cx43 (S369) polyclonal antibodies, SP immunohistochemical method was used. Then SPSS 13.0 statistical software was applied to complete statistical analysis. Results:1. In the normal rat lingual mucosa, the positive staining CK19 cells were distributed in the basal cell layer; mild dysplasia, moderate dysplasia and severe dysplasia displayed CK19 positive staining in cytoplasm of supra-basal layers; In OSCC tissue, CK19 protein was expressed in all the stratum of epithelium.2. The positive rate of CK19 were respectively 30.00%(3/10), 50.00%(7/14),58.33% (7/12), 80.00% (8/10), and 91.67%(11/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05). It proved that the carcinoma lesions develop in tandem with the increasing expression of CK19 and the positive rate.3. In the normal rat oral mucosa, the immunohistological staining of Cx43 protein was mainly in the cell membrane. It was weakly positive in the basal cell layer, increased in the stratum spinosum and stratum granulosum, and negative in the stratum corneum of normal epithelia. Compared with normal epithelia, in dysplastic and cancerous oral epithelia the staining decreased significantly.4.The positive rate of Cx43 were respectively 100.00%(10/10),85.71% (12/14),66.67% (8/12),40.00%(4/10), and 33.33%(4/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05). It proved that Cx43 significantly decreased in normal tongue mucosa, epithelial dysplasia and oral squamous cell carcinoma tissues with the development of lesion degree. Conclusions:1. Expression level of CK19 protein significantly increased with the development of rat tongue carcinoma which is induced by 4NQO, suggesting that CK19 might be related with malignant changes of oral epithelium. The abnormal CK19 expression might be helpful for the early diagnosis of OSCC.2. Expression level of Cx43 protein dramatically decreased with the. development of rat tongue carcinoma which is induced by 4NQO, suggesting that the abnormal expression of Cx43 protein expression is involved in oral mucosa carcinoma origination. Decreasing of Cx43 is an early symptom of oral mucosa carcinogenesis.3. There was inversely correlation between protein expression levels of CK19 and Cx43 in 4NQO-induced rat tongue carcinogenesis. Combining detection of CK19 and Cx43 could improve the specificity and sensitivity of detection in OSCC. |
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