Clinicopathological Significance Of MMP₂ And MMP₉ In Bladder Transitional Carcinoma

PrefaceBladder cancer is the most common malignant tumor of urological system. Recurrence and metastasis are their main biological character-istics. Base - membrane, mainly composed of type IV collagen, is a nature barrier preventing the metastasis of tumor. Type IV collagenase MMP2 ( molecular num of gelatin enzyme A is 72 KD. ) and MMP9 (molecular num of gelatin enzyme B is 92KD. ) , relative to the blad-der cancer, can degrade type IV collagen specifically to promote the metastasis of tumor. Immunohistological method and hybridization in situ were usually used in previous studies about the relation between MMP2, MMP9 and bladder cancer, while the report, in which MMP2 and MMP9 in urine of bladder cancer patients were examined is few. Eymography method was used in our study to detect the expression of MMP2 and MMP9 in the urine of different grade and stage bladder cancer, and the results will be compared with the results of urine shedding cell method to investigate the relation between the level of MMP2, MMP9 in urine and bladder cancer and clarify the value of MMP2 and MMP9 in earlier diagnosis and the selection of operation pa-tern.Materials and methods1. General data40 patients with bladder carcinoma hospitalized from September to November in 2001 were enrolled in this study. There were 30 men and 10 women whose age were from 36 to 77 years old and the mean age was 62 years old. After being confirmed by pathology, these sam-ples were all transitional carcinoma of bladder. According to the standard of WHO, there were 11 cases of grade I , 23 cases of grade II and 6 cases of grade III. According to the standard of UICC, there were 21 cases of stage T₁ , 12 cases f T3 and 7 cases of T3.2. Equipments and reagentsEquipments: electrophoresis apparatus of DYY - III type electrophoresis slot of DYY - III type QUEVE caloristatHomoiothermy cleansing bath of LKB2219 type Overdrive and hypothermia centrifugal machine Reagents; acrylamidemethyleneacrylamidegelatin10% SDS -PAGE glue( including 1 rag/ml gelatin)2.5% Triton X-100Coomassie brilliant blue R2503. Methods(1) Samples collection50 ml morning urine of patients with bladder carcinoma was col-lected and centrifugated for 10 minutes at 3000r/min under 4T!. The deposition was also collected and stored under - 201 waiting for use.At the same time, routine desquamated cells examination was conducted.( 2 ) zymography(1)Samples were defrosted under room temperature and then lyase was added to make homogenate..(2)Cetrifugating for 60 minutes at 15000r/min under 4CC.(3)The upper clear fluid was collected and fixed protein quantity by phenol reagent method. 25#J sample was collected at the same pro-tein concentration and add to 10% SDS - PAGE glue ( polyacrylamide gel) which had l.0mg/ml gelatin. 5% concentrated glue was add to the upper layer as separating glue. Electrophoresis was conducted for 3 hours at 30 mA constant current.(4)After electrophoresis, the gel was soaked in 2. 5% Triton X -100 for 3 hours.(5)Then incubated at 50Mm Tris - Hcl, 200mmol/L NaCl, l0mmol/L CaCl2 fluid whose PH value was 7. 6 for 30 hours.(6)After incubation, stained in 0. 5% coomassie brilliant blue R250, 30% methanol and 10% acetic acid for 4 hours.(7)Discolorated by discoroloration fluid( methanol: acetic acid; water = 3 :1:6) for 4 hours.(3)1 mage analysisThe results of Zymography electrophoresis were gray scanned by UVP gel image analysis system. The gray area represented the activity of enzyme and the enzyme activity of cells was quantitatively com-pared.(4) Statistical analysisThe mean value between two groups were analyzed by t test and the rates between two groups were analyzed by x^?2? test.Results1. The comparison of the activity of MMP2 and MMP9 in the urineof bladder carcinomaThe positive results of MMP2 and MMP9 were the penetrating band under the blue background. 32 cases expressed MMP2 in 40 ca-ses while 38 cases expressed MMP9. 2 cases neither expressed...