| 摘要 |
The method for assaying the content of active and latent forms of gelatinases in the tissues of the patients with varicocele comprises homogenization in 1 % solution of sodium dodecylsulfate, the centrifugation of homogenate 3000 rpm. 20 µL are applied to the gel for electrophoresis at 150 V for 4 hours at the temperature of +. Following electrophoretic separation, the gel is washed in Triton X-100 solution, incubated in the buffer with calcium chloride at pH=7.5 for 18 hours at the temperature of +, fixed and stained with 0.25 % Coumassie brilliant blue. Then the gel is washed in the solution of the acetic acid with methanol. The proteolytic activity of gelatinases is visualized as destained bands on the blue background with their localization corresponding to the molecular mass of each enzyme according to the standards of the molecular masses. The microsurgical subinguinal varicocelectomy is performed under the local anesthesia with Lidocaine and Buivacaine. The skin is incised along longitudinally at the distance of from the external inguinal ring. The fasciae are separated. The spermatic cord is mobilized and fixed onto the hook or rubber holders, immobilized at 8-18 magnification, ligated, and the external seminal vein is cut. Upon opening of the spermatic cord, the veins, the seminal artery and the lymphatic vessels are differentiated intraoperatively with the aid of the complex for Dopplerography. All the veins are separated, ligated, cut, and the tissue specimen (part of the varicose vain of the spermatic cord) is sampled for further examination. The sample is transported to the laboratory at the temperature of liquid nitrogen. The fasciae and subcutaneous fat are sutured layer-by-layer. The skin is sutured with Vicril 4/0. The sample is stored at the temperature of the liquid nitrogen not more than one month. Prior to the assay, the sample is minced in liquid nitrogen. The concentration of active and latent forms of matrix metalloproteinases is measured by zymography in 12 % polyacrylamide gel with sodium dodecyl sulfate and 0.1 % gelatin as a substrate. The areas of lysis are measured and the concentrations of the active forms of enzymes are determined using the standard set of matrix metalloproteinases. |
