The MRNA Expression Patterns, Correlativity And Clinical Significance Of MMP-3,MMP-13,TIMP-1 And TIMP-2 In A Rat Model Of Tongue Squamous Cell Carcinoma

Oral and maxillofacial-head and neck cancer is the sixth largest common cancer over the wold, more than 80% of which are oral and maxillofacial squamous cell carcinoma. Along with the medical science and technology progress and the various treatment means of promotion, tumor local control has been significantly improved. However, in the past 20 years, the 5-year survival rate of the oral cancer remained at 50-55 percent, that there was no obvious improvement. Currently more and more attention were paid to the etiology, diagnosis, treatment and prognosis of oral cancer. With the development of tumor molecular biology, it revealed that malignant tumors were relevant to activation of various oncogene and inactivation of tumor-suppressor gene and apoptosis of regulating disorders. Oncogene activation and tumor-suppressor gene inactivation got involved in the development process of cancers.Tumor,especially malignant tumor has been the hot and difficulty as numerous research scholar's discussion in tumor local invasion and distant metastasia.In the oral cancer,90 percent is the squamous cell carcinoma,of the tongue department, the incidence of squamous cell carcinoma is the highest part. The occurrence and development of the tongue squamous cell carcinoma is a complicated process involving multiple factors. The main reason why the oral cancer has higher incidence and lower cure rate is due to the nature of oral cancer wich is the development and distant metastases. So far we can't explain why the infiltration is so strong in oral cancer. Many medical applicants put forward various tumor metastasis, but the exact theory of molecular biology mechanism is still unknown,the methord of obtaining the ability of infiltration and metastasis as tumor cells as well as penetrating the basement membrane into the blood and lymph in order to reach distant metastasis, also needs to years of efforts to unlock the mysteries.Infiltration and distant metastases mechanism of malignant tumor is.uncl-ear,however,so far as we known is that matrix metalloproteinases(MMP) and tissue inhibitors of MMP(TIMP) as well as various cell factors and growth factors can change the activity of cancer cells.MMPs comprise a family of zinc-dependent proteolytic enzymes, which collectively can degrade most, if not all, components of the basement membrane and ECM with overlapping specificity.They have the similar structure,including 5 different domain,(1)hydrophobic signal peptide sequence;(2) propeptide section;(3) catalytic activity section;(4) rich in proline hinge section;(5)carboxyl terminal section. Propeptide section and catalytic activity section are highly conservative. It has certain substrate specificitybetween various MMP, but not absolutely. Variety of extracellular matrix components can be degraded by the same kind of MMPs, but a certain kind of extracellular matrix components can be degraded by multiple MMP, however different enzyme degradation efficiency can be different. MMPs are almost able to degrade all kinds of protein components of ECM and destroy the histologic barrier of tumor cells in invasion so that they can play a critical role in tumor invasion and metastasis,which is considered the process major proteolytic enzymes, that causes more attention on MMPs. MMPs inhibitors can be divided into natural inhibitors and artificially chemical synthesis ones,Natural inhibitors have tissue inhibitor of matrix metalloproteinases (TIMP) and alpha 2 gigantic globulin. The former was paid more attention between them. Artificial synthetic inhibitors become the new anti-cancer drugs concerns due to they can be owned batch production and have better function in inhibiting tumor infiltration and metastasis. All members of the family of MMPs all can be inhibited by a group of tissue inhibitors of matrix metalloproteinases,TIMPs). Within the body,TIMPs and MMPs keep a balance relationship, that defined the activity of MMPs, it will affect the infiltration and metastasis if the cancer destroy this balance relationship. The study of MMPs and TIMPs are lack in tongue squamous cell carcinoma from the domestic and foreign literature. The present study aimed to explore the expression pattern,correlativity and clinical significance of MMP-3,MMP-13, TIMP-1 and TIMP-2 in a rat model of tongue squamous cell carcinoma. It may provide new materials and clues in occurrence, development, invasion and metastasis of oral tongue squamous cell carcinoma and then provide the help in prevention, diagnosis and treatment for tongue squamous cell carcinoma.Chapter oneObjectsTo demonstrate the mRNA expression of matrix metalloproteinases-3 (MMP-3) and matrix metalloproteinases-13(MMP-13) in the progressive phases of tongue squamous cell carcinoma in rats,and infer the role for clinic.Methods1. The establishment of Sprague-Dawley(SD) rats tongue squamous cell carcinomas transition model:fifty adult female SD rats were housed under specific pathogen-free conditions with controlled light/dark cycles and free access to water and rodent chow.The experimental group was given 4NQO and the control group was given normal saline.2. The SD rats were sacrificed when they grew to about 16 weeks and 24weeks.Then the tongue was divided into two parts. One part of it was fixed with 4% paraformaldehyde and paraffin-embedded, the other part was snap frozen by liquid nitrogen and stored in a -80℃. ultra-refrigerator.3. Observe the organization changes after the paraffin-embedded tissue section was made by HE staining.4. The kinetics of MMP-3 mRNA and MMP-13 mRNA expression wad assayed by fluorescence quantity reverse transcriptase-polymerase chain reaction. 5. Compared the mRNA expression of MMP-3 and MMP-13 in the control group, epithelial dysplasia group and the tongue in squamous cell carcinoma group.6. Analysis of relative gene expression data use the 2-ΔΔCt method,SPSS 13.0 and Microsoft Excel software were used for satistical analyses. One-Way ANOVA was used to determine the significance of differences in means.Results1. HE staining:epithelial basal cell polarity disappears;nuclear and cytoplasm increase;mitotic figures are increased; cellular pleomorphism; hyperchromatic nuclei; nucleoli increase,all of above were found in the epithelial dysplasia.As the squamous cell carcinoma, tissue was composed of a large number of tumor cells. Tumor cells vary in size tightly arranged and there are little intercellular substance, high nucleocytoplasmic ratio, heteromorphism. The nuclear shape and size varied. SD rats animal models were established.2. In normal rat tongue,a faint band for both MMP-3 mRNA and MMP-13 mRNA was observed by fluorescence qRT-PCR. The mRNA expression of both MMP-3 and MMP-13 was increased in the tongue of the rats treatead with 4-nitroquinoline-1-oxide(4NQO) in epithelial dysplasia. And the tongue in squamous cell carcinoma rats showed a marked mRNA expression.3. The analysis results th mRNA of MMP-3 and MMP-13 showed that difference among groups was significant. For MMP-3 mRNA, The difference was statis-tical significant compared normal group and epithelial dysplasia,also was statistical significant compared normal group and the tongue in squamous cell carcinoma rats,the same to the epithelial dysplasia and the tongue in squamous cell carcinoma.For MMP-13 mRNA, the difference was statistical significant compared normal group and epithelial dysplasia,and it was statistical significant between the tongue in squamous cell carcinoma and normal group and the epithelial dysplasia.Chapter twoObjectsTo explore the mRNA expression of tissue inhibitors of matrix metalloprotein- ases-1 (TIMP-1) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) in the progressive phases of tongue squamous cell carcinoma in rats,and conjecture the guiding significance and value for clinic.Methods1. The establishment of Sprague-Dawley(SD) rats tongue squamous cell carcinomas transition model:fifty adult female SD rats were housed under specific pathogen-free conditions with controlled light/dark cycles and free access to water and rodent chow.The experimental group was given 4NQ0 and the control group was given normal saline.2. The SD rats were sacrificed when they grew to about 16 weeks and 24 week-s.Then the tongue was divided into two parts. A part of it was fixed with 4% paraformaldehyde and paraffin-embedded, the other part was snap frozen by liquid nitrogen and stored in a -80℃. ultra-refrigerator.3. Observe the organization changes after the paraffin-embedded tissue section was made by HE staining.4. The kinetics of TIMP-1 mRNA and TIMP-2 mRNA expression wad assayed by fluorescence quantity reverse transcriptase-polymerase chain reaction(qRT-PCR).5. Compared the mRNA expression of TIMP-1 and TIMP-2 in the epithelial dysplasia and the tongue in squamous cell carcinoma with the control group.6. Analysis of relative gene expression data use the 2-ΔΔCt method,SPSS 13.0 and Microsoft Excel software were used for satistical analyses. One-Way ANOVA was used to determine the significance of differences in means.Results1. HE staining:Changes in the epithelial dysplasia were the same to the ones in chapter one. And the pathological lesions in the tongue squamous cell carcinoma were the same as those in chapter one.2. In normal rat tongue,a faint band for TIMP-2 mRNA and following is stronger and stronger in the epithelial dysplasia and the tongue in squamous cell carcinoma respectively. And for TIMP-1 mRNA,the least expression is in the normal,and the most expression is in the squamous cell carcinoma,between them is the epithelial dysplasia.3. Observe the organization changes after the paraffin-embedded tissue section was made by HE staining.4. The analysis results of TIMP-1 and TIMP-2 showed that difference among groups was significant.The result showed that both the TIMP-1 and TIMP-2 were statistical significant compared normal group and epithelial dysplasia,and was statistical significant compared normal group and the squamous cell carcino-ma,also was statistical significant compared the squamous cell carcinoma and epithelial dysplasia.Chapter threeObjectsTo study the correlation relationship of the mRNA expression of MMP-3,MMP-13 and TIMP-1,TIMP-2 in SD rats model of tongue squamous cell carcinoma, and understand the the guiding significance and value for clinic.Methods1. The establishment of Sprague-Dawley(SD) rats tongue squamous cell carcinomas transition model was the same to that in chapters before.2. The SD rats were sacrificed when they grew to about 16 weeks and 24 weeks.Then the tongue was divided into two parts. A part of it was fixed with 4% paraformaldehyde and paraffin-embedded, the other part was snap frozen by liquid nitrogen and stored in a -80℃. ultra-refrigerator.3. Observe the organization changes after the paraffin-embedded tissue section was made by HE staining.4. The kinetics of MMP-3mRNA,MMP-13mRNA,TIMP-1mRNA and TIMP-2 mRNA expression wad assayed by fluorescence qRT-PCR.5. Analysis of relative gene expression data use the 2-ΔΔCt method, infer the related relationship of the mRNA expression of MMP-3,MMP-13 and TIMP-1,TIMP-2, SPSS 13.0 was used for satistical analyses.Bivariate Correlate was used to determine the significance of differences in related relationship. ResultsThe SD rat model of tongue squamous cell carcinoma and changes of tissue with HE staining as well as the mRNA expression of MMP-3,MMP-13 andTIMP-1,TIMP-2 were close to those in chapter one and two.The related relationship.of the mRNA expression of MMP-3,MMP-13 andTIMP-1,TIMP-2 is significant from the mornal group to the squamous cell carcinoma.Conclusion1. The mRNA expression of MMP-3 and MMP-13 was increased in a Rat model of tongue squamous cell carcinoma.2. The mRNA expression of TIMP-1 and TIMP-2 was increased in a Rat model of tongue squamous cell carcinoma.3. The related relationship.of the mRNA of MMP-3,MMP-13 andTIMP-1,TIMP-2 is significant from the mornal group to the squamous cell carcinoma.