The Primary Study Of The Relationship Between Cyclooxygenase-2 And Oral Squamous Cell Carcinoma

Among the oral and maxillofacial malignant tumors, oral squamous cell carcinoma is the most common (about 80% ), with high incidence and low survival rate and danger to human's health. The research expresses, the occurrence and development of oral squamous cell carcinoma has not only excessive propagate of cell, but also repress of apoptosis of cell. The balance of apoptosis and propagate is out of moderate, that is more important in the occurrence and development of the cancer. Cyclooxygenase( COX) is a important rate - limiting enzyme in the synthesize process of the prostaglandin ( PG). It can make arachidonic acid metabolist to all kinds of PG s outcome. And it joins into the process of pathologic physiology of the body. The body can be induced express by the pathologic appearance of the inflammation and tumor. The research of recent years expresses that COX -2 acting as the induced COX besides its importance in inflammation, has something with occurrence and development of many tumors. One way of the COX - 2 to develop the occurrence of the turner is that the downstream PGF2 up - regulates the expression of the vascular endothelial growth factor ( VEGF) , then improves the production of the newborn vascular of the tumor. VEGF is a highly special vascular endothelial cell factor. Combining with its special receiver (VEGFR) , it gives rise to a series of massages transmission ; sends many cell and growth factors; stimulates the proliferation and migration of the vascular endothelial cell and improves the production of the newborn vascular. It is more important in the development and transference of the tumor.Its first that the test uses COX -2 depressant NS - 398 to handle squamouscell carcinoma of human's tongue, and study the function of resisting propagate out of body and the influence of period of the cell propagate. And detect the adjust function of COX - 2 in the course of cell apoptosis and blood vessel making of oral squamous cell carcinoma. To discuss the relation of COX - 2 and oral squamous cell carcinoma, and study the pathogenesis of oral squamous cell carcinoma further, offer theory gist, offer new thought for finding more effect treatment measure.Materials and Methods1. Immunohistochemical staining: A total of 45 oral squamous cell carcinoma tissue specimens, 35 leukoplakia and 12 normal tissues were collected. I use the method of immunohistochemical staining S - P to observe the change of the C0X-2andVEGF.2. The content expression of VEGF mRNA and protein; We detected the content change of VEGF mRNA and protein in the Tac8113 cells of human tongue squamous cell carcinoma which were handled by 150 jxmol/L NS -398.3. Cells culture; Tac8113 cells were cultured in the culture medium with 10% embryo cow blood serum in 37t, 5%CO2 saturation humidity box.4. MTT detect measure; Tca8113 cells were inoculate into the culture board with 0. 5 x 10 /hole. The culture fruid consistence; the culture fruid contains with 0,25,50,75,100,125,150,200|xmol/L NS - 398, which contrast to the culture medium cells with 0.1% DMSO. Each hole joins in MTT solution (5 nig /ml) reagent 20 ul, to culture 4 hours at 37X!, and stop cultureing. Afterward each hole joins in DMSO 150jjj, concusing 10 minutes, we use the detect - machine to detect the OD of each hole.Restrain Rate(% ) : [ 1 - OD( the disposal team)/OD( the contrast team) x 100% ]5. Cell morphology observation(1) Inverted phase contrast microscopeWith the culture fluid which the concentration of NS - 398 is 0 or 150ujnol/L, after48h, 72 h, 96h culture, observing the Cell morphology andtake photos.(2) Transmission electron microscope (TEM)The cells were centrifugated at 4t for 6min at lOOOrpm, 2. 5% amyldialde-hyde fixation, absewed by TEM.6. Determination the phases of cells by flow cytometry.To Inoculate the cells in 6 - holes culture medium by 1 x 106/hole, after 48 - 96 h culture in culture fluid which the concentration of NS - 398 is 0 or 150jxmol/L, together cells growing along the wall, and to make monocellular suspension, washing in PBS two times, and then add into propidium iodide ( PI) solution, reaction for 20 - 30min without light, determinate the phases of cells by flow cytometry. And analyse the result by software ( ModFit LT 3. 0) .7.RT-PCRAfter 72 h culture, to measure the change of expression of apoptosis gene -Bel - 2, Bax, Survivin mRNA, to measure the change of expression of apoptosis gene -Bel-2, Bax, Surviving mRNA.8. Western - blotting analysisQuantitate the protein using Mars method. And measure the change of expression of apoptosis gene - Bel - 2, Bax, Survivin, mRNA in to measure the change of expression of apoptosis gene -Bel-2, Bax, Surviving, mRNA.Experimental Results1. The expression of COX - 2, VEGF in squamous cell carcinoma and leukoplakia :COX - 2 and VEGF both express in the cell plasma and membrane of oral squamous cell carcinoma and leukoplakia. The expression was significantly higher in the most differentiation squamous cell carcinoma than the midding differentiation, the lower differentiation squamous cell carcinoma and leukoplakia. It expressed slight in the normal oral mucous membrane(F = 147.53 ,P <0.001).2. The content expression of VEGF mRNA and Protein:Tca8113 VEGF mRNA and protein in human tongue squamous cell carcinoma by 150 u mol/L NS - 398 gradually descend with the effect time prolonging(F=23.26,P<0.001;F=99.52,P<0.0001).3. Cell culcureThe shape of Tca8113 cell has not regular and the clearance has gradually became wider, volume smaller by NS -398 along with the time prolonging and the consistence increasing.4.MTTThe Inhibition effect of NS -398 to Tca8113 is dose - dependent and time -dependent. (F = 16. 88,P <0.05;F = 199.44,P <0.001).5. Transmission electron microscopeTypical features of apoptosis cells can be seen after reaction with NS - 398 for 72 h under transmission electron microscope: aggregation and concentration of chromatin.6. Determination the phases of cells by flow cytometry.NS -398 can produce large increase of cells in G0/Gjphase, proportionalities of cells in S and G2/M phases are decreased, blockage cells are growing in Go/G! phase(P<0.001).7.RT-PCRAlong with the increase of the reaction time, the expression of mRNA of apoptosis gene Bel - 2 and Survivin are inducing, however, the expression of Bax are on the contrary, the difference is SignificantP <0.001).8. Western - blotting analysisAlong with the increase of the reaction time, the expression of mRNA of apoptosis gene Bel - 2 and Survivin are inducing, however, the expression of Bax are on the contrary, the difference is significant(P <0.001).Conclusions1. COX - 2 and VEGF are highly expressed in early stage of oral mucous membrane carcinoma, and having significant synergistic effect.2. The expression of COX -2 and VEGF in oral mucous membrane carcino-mar is obviously positive relativity.3. In the vitro, expression degree of mRNA and protein of VEGF in Oralsquamous carcinoma cells can be decreased by inhibitor of COX -2 - NS -398.4. Selective inhibitor of COX -2 - NS -398 can inhibit proliferation of cell strain of oral tongue squamous carcinoma - Tca8113 culture in vitro, and is dose - dependent and time - dependent.5. Selective inhibitor of COX - 2 - NS - 398 can block the grow of cell strain of oral tongue squamous carcinoma - Tca8113 at GQ/GX phase in vitro.6. Selective inhibitor of COX -2-----NS -398 can induce the apoptosis oforal tongue squamous carcinoma cells — Tca8113 in vitro.7. Inhibitor of COX -2 - NS -398 can down - regulate the expression degree of Bel - 2 and Surviving in cell strain of oral tongue squamous carcinoma, up - regulate the expression of Bax in cell strain of oral tongue squamous carcinoma - Tca8113, mRNA and protein culture in vitro, and appears time -dependent in some extent.