Matrix Metalloproteinase-2 And Its Tissue Inhibitor In Colorectal Carcinoma

Objective: Colorectal carcinoma is one of the most common malignant tumors in our country. The tendency of incidence is increasing today. Although therapy methods have been improved, survival rate of five years is still very low. Tumor invasion and metastasis not only are the most important cause of death, but also bring patients a lot of distress. So the study for mechanism of invasion and metastasis of colorectal carcinoma has become a focus at present. Many studies displayed that degradation of extracellular matrix (ECM) was the capital step in the process of tumor invasion and metastasis. Matrix metalloproteinases (MMPs) is one of the important proteinases that can degrade all the protein components of ECM. By degrading ECM, MMPs can regulate the biological behavior of tumor cells, such as invasion, metastasis and so on. The high expression and activities of MMPs have been found in many kinds of tumors so far. On the other hand, there is a kind of tissue inhibitor of metalloproteinases (TIMPs) in vivo, which can inhibit the activities of MMPs and hold back the invasion and metastasis of carcinoma. The present study aimed at finding the expressive characteristic of MMP-2 and TIMP-2 and the relationship between them in colorectal carcinoma tissues using gelatin-zymography assay, immunohistochemistry staining and Western blotting, investigating the ratio of MMP-2/TIMP-2 in tumous development and it's clinical values.Methods 1 Specimens: 25 cases of colorectal carcinoma specimens and paired normal tissues were collected from patients operated in the Surgery Department of the Fourth Hospital of Hebei Medical University during March 2002 to July 2002. All specimens were verified by pathology. The distance from carcinoma to normal tissue is at least 10 centimeters. All specimens were collected within half an hour after the operation and stored at –70℃.2 Activity of MMP-2 assay: Gelatin-zymography assay was used to detect the activity of MMP-2 in normal and colorectal carcinoma tissues. The results were imported into the computer and analyzed by 1D digital imaging system.3 Western blotting of MMP-2 and TIMP-2: The extracts from normal and colorectal carcinoma tissues were separated on 8%SDS-PAGE, and then blotted onto nitrocellulose membrane. The membrane was immunologically stained with anti-MMP-2 and anti-TIMP-2 antibody. The results were analyzed by digital imaging system. 4 Immunohistochemistry staining was used to detect the expression and distribution of MMP-2 and TIMP-2 in normal and colorectal carcinoma tissues. The experiment procedures were according to the Test Kit introduction. The paraffin sections were incubated with ployclonal antibody of anti-MMP-2 and anti-TIMP-2( 1:200 dilution ).Results1 Activity of MMP-2 in normal and colorectal carcinoma tissues: the results of gelatin-zymography assay showed that the MMP-2 was the major gelatinase in the specimens, including two forms: pro-MMP-2 and MMP-2. The activity of pro-MMP-2 and MMP-2 was detected in both normal tissues and colorectal carcinoma issues, but the activity of colorectal carcinoma tissues was much higher than that of the normal tissues. There was a significant difference between them (P<0.01). The activity of pro-MMP-2 and MMP-2 in the colorectal carcinoma tissues with serous membrane layer or surrounding soft tissue invasion was much higher than that of the carcinoma tissues with muscular layer invasion. There was a significant difference (P<0.05) between them. The activity of pro-MMP-2 and MMP-2 in the colorectal carcinoma with lymph node metastasis was much higher than that of the colorectal carcinoma tissues without node metastasis. There was a significant difference (P<0.01) between them. Moreover, with the progression of Dukes stages, the activity of pro-MMP-2 and MMP-2 was gradually increased. 2 Western blotting suggested that the level of MMP-2 in colorectal carcinoma tissues was obviously higher than that of the normal tissues and the content of TIMP-2 in colorectal carcinoma tissues was much lowe...