Studies Of Application For FCM And AgNORs On Palatal Carcinogenesis In Wistar Rats

Backgrond:Oral cancer is the most common malignant tumors in the oral and maxillofacial, which is the sixth tumor of whole body malignant tumors. While the largest number of oral cancer is squamous cell carcinoma (OSCC), at least 90%. The OSCC's development is a gradual process. Histologically, from normal oral mucosa to oral cancer, the process is epithelial hyperplasia, epithelial dysplasia, cancer in situ, and last is infiltrating carcinoma.When the patients visit it has already been advangced. Although it can be treated by surgery, chemotherapy and radiation therapy, and so on, it will have caused irreparable damagefor patients, as damage oral and maxillofacial organization and function, and do serious harm to the patient's physical and mental health. Therefore, how to detect oral mucosal abnormalities early and make treatment for the change of mucosa is especially important, and it is a major focus on research of oral cancer.Flow cytometry, originated in the 20th century 70s, is a new technology for single-cell quantitative analysis.FCM is most widely used for the areas of immunology, hematology, oncology and cell apoptosis and so on.FCM can measure DNA content and detect aneuploid malignant cells. It need to spit the tumor tissues, prepare single cell suspension, then embed DNA with fluorescent dye in order to develop DNA, and at last, analyse DNA content on the computer. Normal somatic cells are diploid, while tumors often appear aneuploid, including tetraploid and aneuploid. In tumor, Chromosome's number and structure would be abnormal, that would lead to uncontrolled cell growth.This is an important feature of malignant tumors. It reported that the accuracy rate of diagnosis for malignancies by FCM was as high as 90%.One sign of malignant is that cells proliferate indefinitely. Nucleolar organizing region (NOR) is a DNA fragment located on chromosome, containing the rRNA gene, that participate in the formation of nuclei and is a sign of rRNA transcriptional activity. It can reflect the changes of nucleus' structure and function.NOR contains the nucleolar organizer region associated proteins, combining with silver ions and color developping. Then we will estimate the state of cell proliferation well though by the computer.Objectives:In this study, oral cancer model was induced by chemically method. Diagnose the lesion combining with FCM and AgNOR for, then evaluate the two methods.We should find a more effective method for detecting oral cancer, to lay the groundwork for early diagnosis for human oral cancer.Methods:1. The establishment of animal model: 63 Wistar rats of experimental group were divided equally, each cage 7 and total 9 cages. Smear the 0.5% 4NQO solution on the rat's palate mucosa with a cotton swab, once every other day, and continue 28 weeks. In control group do nothing but conventional water diet was used. From beginning of the experiment, at fhe 4^th,8^th,12^th,16^th,20^th,24^th,28^th week we killed 9 rats in the treatment group and 1 in the control group each time.2. In the process of establishing model, since the 4^th week, we brush the suspicious lesion of the rats' palate before killing them to collect the cells and prepare the cell suspension, which would be measured by FCM.And then following procedure was: killed them, cut the palate, fixed them into formalin solution, dehydrated with gradient alcoho, embedded with paraffin, conventional slice, stained with HE at last, sealed with Canada gum. Observed under the microscope. Afterward, take another slice to make a AgNO₃ staining, in order to count the number of AgNORs.3. Judging:Criterion of FCM: Normal rat palatal oral mucosa is a reference, whose cell DNA is diploid, and the DI is 1 .0.We can obtained the DI of the experimental group from computer software. The result out of the range of 1.0±0.1 indicates that the of aneuploidy automatically.Criterion of oral mucosal lesions in rats:Pathological changes of rat oral mucosa including epithelial dysplasia and oral cancer refer to WHO criterion(1996,1997).Criterion of AgNORs:It refers to standardization of reserch program about AgNOR, November 1995, counting the total number of particles. Then translated it into an average of AgNOR per nucleus. While observe the nuclear particle's morphology.4. We analyzed the results using ANOVA and considered statistieally significant level as a=0.05. Then make linear correlation analysis about the two methods.Result:The establishment of animal model:With the extension of time for smearing,rat's palatal mucosa have produced a white thickening, granular hyperplasia, ulceration, erosion and other changes.At 4th weeks all the changes were epithelial hyperplasia in rats.At 8th week 55.6% were the simple hyperplasia,44.4% were mild dysplasia.At 12th week 22.2% were mild dysplasia,55.6% were moderate dysplasia,22.2% were severe dysplasia. At the 16th weeks 44.4% were moderate dysplasia, and 55.6% were severe dysplasia.At the 20th week 11.1% were moderate dysplasia,77.8% were severe dysplasias, and 11.1% were the carcinoma in situ; At the 24th week,55.6% were severe dysplasia and 33.3% were carcinoma in situ,11.1% were squamous cell carcinoma; At the 28 week,22.2% were carcinoma in situ,77.8% were squamous cell carcinoma.1. It has a highly sensitive diagnosis about moderate and severe dysplasia, carcinoma in situ and squamous cell carcinoma with FCM for 78.6%. It has positive relationship between FCM and AgNORs.Conclusions:1.4NQO solution can be successfully used to smear on the Wistar rat palatal mucosa in the establishment of animal model of oral cancer,whose process of carcinogenesis is similar to human.Mouse palatal careinogenesis was a proeess which experienced a squamous cell hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia and carcinoma in situ, furtherly to early invasive careinoma.2. To measure DNA content by FCM and AgNORs can be used as an adjunct to oral cancer diagnosis. It can improve the detection rate of oral mucosal lesions by combining FCM with AgNORs.