| Tongue cancer is a common malign tumour in head and neck. Because of its plentiful lymph and blood supply, lymphatic metastasis is easy to occur in the early period. Although we have studied a lot about the causes, development, clinical outcomes and treatment methods of tongue cancer, we are not sure about its mechanism, and its clinical effects need improving. Aiming at the preventive methods of malign tumours in head and neck, including tongue cancer, exploring effective treatment means and improving patients' survival rate are always the emphasis of our study. The study shows that the occurrence of tongue cancer is closely related to the existence of oral epithelial dysplasia and its canceration. Clinical epithelial dysplasia includes oral leukoplakia, erythroplakia and human papilloma, which are dangerous factors of oral cancers. Their common characteristic is epithelial dysplasia in pathology.The formation of oral cancers is affected by all kinds of hereditary factors, such as the change of cancer genes and cancer suppressor gene. From epithelial dysplasia to its canceration, there is the participation of a large number of related molecules, which include apoptosis-modulating abnormality, abnormal DNA expression and the expression changes of many tissue biomarkers. COX-2 was discovered in recent years, and it plays an important role in the expression abnormality closely related with the occurrence and development of malign tumours. It is a focus to prevent and cure malignant tumors by choosing COX-2 inhibitors to inhibit COX-2's activity. The experiments and clinical observations show that COX-2 inhibitors can prevent and cure solid malignant tumors, increase the sensibility of radiation treatment and chemo-treatment as an adjuvant therapy, and reduce gastrointestinal complications. Therefore, it can remind us of the possibility to use COX-2 inhibitors to intervene the canceration of leukoplakia in oral precancerosis, and it is very important for the prevention of oral cancers.My research is aimed at the relativity of COX-2 and oral mucous membrane's precancerous condition pathological changes and oral cancers' outbreak, as well as the characteristic of the highly assimilation about local application in mucous membrane. We set up SD rats tongue cancer models by DMBA's chemical induction. And we also locally use COX-2 inhibitor Celecoxib to intervene. We study the local application of selective COX-2 inhibitor Celecoxib to inhibit the occurrence and canceration of oral epithelial dysplasia. We detect the expression of proapoptotic protein caspase-3, Survivin in the formation of tongue cancer induced by DMBA, and explain the effect of COX-2 inhibitors on promoting apoptosis in order to provide favorable references for further developing the methods of mainly inhibiting COX-2 enzyme activity and local application to prevent leukoplakia's occurrence and canceration.80 rats are randomly divided into two groups,40 in control group and 40 in experimental group. The control group (Group A) is the tongue cancer pure modeling group, which is divided into A1, A2, A3 and A4, with 10 rats in each group. After the animals are under anaesthetic, sand paper is used to clean the experimental rats's periglottis in order to make it hematose but not errhysis. And we will use a super-thin pen to smear 1% 7,12-DMBA acetone in their lingual margins, three times a week. The experimental group (Group B) is local application group, which is divided into B1, B2, B3 and B4, with 10 rats in each group. We will give them the same carcinogen treatment; at the same time, we will give them 6% Celecoxib cataplasm (provided by Traditional Chinese Medicine of South Medical University), once a day. We will observe each rats's living condition, weight change, tumor formation rate. They will be respectively killed on the 8,12,16 and 20 week,10 rats for each time and each group. After they have been killed, we will cut their whole bodies of tongue and divide them into two parts. And we will get 500mg fresh tissues in each group and keep them in -80℃in the refrigerator; the rest is kept in 10% formalin. We will observe the changes of periglottis in the process of SD rats induced by DMBA through light microscope; and we will analyze by using image-pro plus software, measure the stratum thickness of each sample. Then we will choose 10 points on the epidermis of each sample and get the average result after we measure each one. We use immunohistochemical assay and Western blot to detect caspase-3, Survivin protein expression, and we will use RT-PCR to detect the m-RNA's horizontal variations of caspase-3 and Survivin.The results of my study show that tumors don't take place both in the control group and in the experimental group in the first 8 groups. The canceration rate of the control group is respectively 20%,60% and 70% in the 12th,16th and 20th week, while there are only two mice have periglottis canceration in the 20th week, and its canceration rate is 20%. The canceration rate of the control group in the whole process is 37.5, while the experimental group is 5%. The difference between two groups has some significance in statistics (p=0.000). Trough the light microscope, we find that with DMBA stimulating time increasing, the thickness of epidermis gradually increases in the control and experiential group. The results of detecting the average thickness of epidermis show that the thickness of epidermis in the control group is respectively (77.30±21.80)μm(63.80±22.10)μm,(88.30±31.70)μm and (95.00±36.20)μm. Compared with the control group, the increasing degree in the experimental group is small (p=0.012); in the comparison of the thickness of epidermis abnormal pathological changes, there is no clear difference between the control group and the experimental group (p=0.052). Both of the two groups don't show that drug intervene has some effects on abnormal increasing thickness. In the comparison of the thickness of keratin, it also shows that the increasing degree in the experimental group is smaller than the control group (p=0.000). In the comparison of inflammation soakage, with DMBA stimulating time increasing, the degree of inflammation soakage increases. The inflammation cells in element areas (5184 um2) are 6.77±2.36,10.74±10.02,17.68±13.20å'Œ17.22±13.88 in the 8th,12th,16th and 20th week, while The inflammation cells in element areas in the experimental group are 9.30±2.59,10.04±3.01,9.02±2.08å'Œ9.70±3.36. Compared with the control group, the degree of inflammation soakage in the experimental group is low, and the difference is significant in statistics (p=0.047).The result of the method of immunohistochemistry assay show that caspase-3 expression in the control group decreases with DMBA stimulating time increasing. The average expression rates are (61.36±6.16)%,(51.18±10.97)%,(48.11±9.57)% and (39.73±11.07)% in 8th,12th,16th and 20th week. The difference is significant in statistics (p=0.000), While caspase-3 in the experimental group has no clear change. The average expression rates are (75.23±7.00)%,(76.75±7.39)%,(79.83±8.56)% and (71.66±16.57)%. The difference is insignificant in statistics (p=0.394). The expression rate of the whole experimental is obviously higher than that of the control group, and the difference between two groups has significance in statistics (p=0.000). Survivin expression rate gradually increase with DMBA acting time increasing. The average expression rates are (12.45±6.07)%,(26.50±20.16)%,(46.33±24.97)% and 58.49±35.94% in 8th,12th,16th and 20th week. The difference is significant in statistics (p=0.000), while the experimental group has no clear change. The average expression rates are (6.66±3.88)%,(6.45±3.45)%,(5.76±3.87)% and (11.51±12.20)%. The difference is insignificant in statistics (p=0.242). The expression rate in the experimental is lower than that in the control group, and the difference between two groups has significance in statistics (p=0.000). The analysis of Western blot also proves that caspase-3 expression in the control group decreases with DMBA stimulating time increasing, and its expression difference in different time has significance in statistics (p=0.000), while caspase-3 expression in the experimental group doesn't change with DMBA stimulating time increasing. Its expression difference in different time has significance in statistics (p=0.000). The analysis of Western blot also proves that Survivin expression in the control group decreases with DMBA stimulating time increasing, and its expression difference in different time has significance in statistics (p=0.000). Although Survivin expression in the experimental group increases with DMBA stimulating time increasing, and its expression difference in different time has significance in statistics (p=0.000), its expression intensity is obviously lower than that in the control group, and the difference between two groups has no significance in statistics (p=0.000).The RT-PCR results show that the expression of caspase-3 mRNA in the control group decreases with DMBA acting time increasing. The expression levels are 0.73±0.12,0.68±0.18,0.48±0.22 and 0.39±0.22 in 8th,12th,16th and 20th week. The expression difference in different time has no significance in statistics (p=0.095). The expression of caspase-3 mRNA in the experimental group is clearly higher than that in the control group, and the difference between two groups has significance in statistics (p=0.004). The expression levels of Survivin mRNA in the control group are 0.57±0.12,0.72±0.17,0.77±0.15 and 0.80±0.11 in 8th,12th,16th and 20th week, and its expression difference in different time has significance in statistics (p=0.000), while the expression levels of Survivin mRNA in the experimental group have no clear change. They are 0.29±0.10,0.26±0.10,0.36±0.09 and 0.39±0.11, and its expression difference in different time has no significance in statistics (p=0.125). The Survivin mRNA expression level of the whole experimental group is lower than that of the control group, and the difference is significant in statistics (p=0.000).In summary, we confirmed that topically administration of Celecoxib paste could inhibit DMBA-induced SD rat tongue tissue epithelial dysplasia and canceration. The mechanism may be correlated with the regulation of the expression of caspase-3,survivin protein and mRNA related to apoptosis. It provides experimental basis for further studying COX-2, the formation of tongue cancer, and the usage of COX-2 inhibitors to prevent oral precancerous condition's canceration and the occurrence of tongue cancer. |
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