| Colorectal carcinoma is one of the most common malignant tumors with a relatively high incidence in China. Despite major advances in the diagnosis and treatment of this disease, its mortality has remained unchanged during the last 20 years. Tumor invasion and metastasis are considered to be the major causes of death in colorectal carcinoma patients. Recent researches in the field of mechanism for tumor invasion and metastasis have demonstrated that the degradation of extracellular matrix (ECM) and basement membrane (BM) is an essential step and the contribution of matrix metalloproteinases (MMPs) is very important during this process. MMPs are a family of zinc-dependent endopeptidases that are collectively capable of degrading most components of the basement membrane and ECM. Several studies have proved that there are high expression level and activity of MMPs in many kinds of tumors, such as carcinoma of esophagus, lung, stomach, etc. Tissue inhibitors of metalloproteinases (TIMPs), as the main endogenous inhibitors of the metalloproteinases, can reversibly inhibit MMPs in a 1:1 stoichiometric fashion and influence the process of tumor invasion and metastasis. At present, there have been many reports about the overexpression of MMP-2 in colorectal carcinoma tissues. However, few reports concerning the detailed pathophysiological significance of MMP-2 and the relationship between MMP-2 and TIMP-2 are available. Therefore, the aim of the present study was to investigate the expression characteristics of MMP-2 and TIMP-2 in colorectal carcinoma tissues and to explore the relationship between MMP-2 and TIMP-2 and the correlation with colorectal carcinoma progression, trying to provide a valuable marker for its clinical prognosis. 1.Change of β-D-Galactosidase and MMP-9 Activity in Digestive Tract Objective: To investigate the differential profile of β-D-galactosidase (GAL) activity in digestive tract mucosa tissues and the change of GAL levels in tumor tissues we established a simple method to measure GAL activity, and detected GAL activity in various digestive tract mucosa tissues. Methods: 1.Establishment of method for GAL activity assay (1)Preparation for enzyme extract: The digestive tract mucosa tissues were washed with phosphate buffer (10mmol/L, pH6.5), minced and homogenized. The enzyme extracts were collected by centrifugation. The protein concentration was determined by modified Lowry assay using bovine serum albumin as standard. (2) GAL activity assay: The final concentrations of components in 1ml of reaction mixtures were as follows: 0.8μmol o-nitrophenylβ-D-galactopyranoside (ONPG) in 0.1mol/L citrate buffer (pH4.5) and 2mg protein of enzyme extract. After incubation for 30 min. at 37oC, the reactions were stopped by addition of 1ml 0.5mol/L sodium carbonate. The absorbances at 405nm were read. Enzyme activity was expressed in U/mg protein. 2.Changes of GAL activity in digestive tract carcinoma tissues 31 cases with digestive tract tumors operated were selected, in which 8 cases were rectal adenocarcinomas, 6 cases were colonic adenocarcinomas, 6 cases were cardiac adenocarcinoma, 5 cases were gastric adenocarcinoma, 6 cases were esophageal squamous carcinoma. Assays of GAL activity in mucosa enzyme extract were performed using the method. 3.MMP-9 relative activity assay in digestive tract mucosa tissues MMP-9 relative activity was detected in rectum (6cases), colon (5cases), cardiac (6 cases), gastric(5 cases), esophagus (6 cases) of the normal and the tumor tissues using gelatin-zymography assay according to previously described. Results:1.Establishment of method for GAL activity assay The GAL activity was measured using this method. The results showed that the variability within batchs was 3.92%～4.89% and the variability between batchs was 3.8%. These suggested that the method was so reliable that it could be used in GAL assay. The method had the merits of high precision, good reproducibility and was easy to operate, so it could detect GAL activity successfully. 2.Changes of GAL activity in digestive tract carcinoma mucosa The results showed that GAL activity could be detected in digestive tract normal mucosa tissues and it was higher in colon (0.47±0.05)-rectal (0.43±0.03) mucosa than that in other mucosa tissues. In the cases of colon (0.60±0.06)-rectal (0.77±0.07) carcinoma group the GAL activity was significantly higher than that of the normal (P<0.05). 3.Changes of MMP-9 relative activity in digestive tract tumor tissues The results suggested that extract of digestive tract carcinoma tissues mainly contained 92kD gelatinase B (MMP-9). MMP-9 relative activity in any digestive tract tissue of the carcinima group (colon 88.00±2.72> esophagus 64.17±6.90> cardia 64.00±2.73 >gastric 50.80±5.20 >rectum 46.00±3.18) was much higher than that of the normal mucosa (cardia 52.00±2.49> colon 50.80±8.98> rectum 38.33±2.04> gastric 31.00±3.65> esophagus 26.67±7.19)(P<0.05). Conclusion: The results suggested that GAL and MMP-9 activities could be detected in digestive tract normal mucosa. It might be correlated with the digestion and absorption of carbohydrate and the renew activation of glandular cell. Their activities in tumor tissues were significantly higher than that of the normal. According to the fact that GAL and MMP-9 play key roles in the modification of cell surface carbohydrate composition and the remodelling of extracellular matrix (ECM), we concluded that they might be involved in the invasion and metastasis of digestive tract carcinoma.2.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 ). Results: 1.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 colorectalcarcinoma tissues was obviously higher than that of the normal tissues and the content of TIMP-2 in colorectal carcinoma tissues was much lower than that of normal tissues. There was a significant difference (P<0.01). The ratio of MMP-2/TIMP-2 in colorectal carcinoma was much higher than that of the normal tissues. There was a significant difference between them (P<0.01). The expression levels of MMP-2 and TIMP-2 in colorectal carcinoma tissues with serous membrane layer or surrounding soft tissue invasion were both higher than those of the carcinoma tissues with muscular layer invasion. There was a significant difference (P<0.05) between them. The expression levels of MMP-2 and TIMP-2 in colorectal carcinoma tissues with lymph node metastasis were both much higher than those of the colorectal carcinoma tissues without lymph node metastasis. There was a significant difference (P<0.01) between them. With the progression of Dukes stage, the contents of MMP-2 and TIMP-2 were gradually increased and the ratio of MMP-2/TIMP-2 was gradually declined. 3.The results of immunohistochemistry staining suggested that there were more positive particles in colorectal carcinoma tissues than that of normal tissues. The positive particles of TIMP-2 in normal tissues were much more than those of colorectal carcinoma tissues. The positive particles of MMP-2 were mainly distributed at the cytoplasm and surroundings of carcinoma cells. TIMP-2 was mainly distributed around the carcinoma. These results were consistent with the results of gelatin-zymography assay and Western blotting. Conclusions: 1.The expression level and activity of MMP-2 in colorectal carcinoma were significantly increased compared with normal tissues and both of them were related to the invasion depth, lymph node metastasis and the Dukes stage. The results suggested that MMP-2 might play an important role in the process of invasion and metastasis of colorectal carcinoma. 2.The expression level of TIMP-2 in colorectal tissues was significantly lower than that in normal tissues. With the progression of invasion depth,lymph node metastasis and Dukes stage, the expression of TIMP-2 gradually increased. This showed that TIMP-2 might be useful for monitoring invasion and metastasis in colorectal carcinoma. 3.The ratio of MMP-2/TIMP-2 in the colorectal carcinoma was gradually declined from Dukes A to D stage, suggesting TIMP-2 increased at later stages of tumor. Thus, for prejudging the colorectal carcinoma invasion and metastasis, TIMP-2 might be of a greater value. 3.Osteopontin signaling in progression of colorectal carcinoma Objective: To observe expressive pattern of osteopontin (OPN), integrin β3, focal adhesion kinase(FAK), phosphatidylinositol 3 kinase(PI3K) and the relationship between them and the progression of colorectal carcinoma, investigate the signal pathway mediated colorectal carcinoma invasion and metastasis. Methods: Expression of OPN, β3, FAK, PI3K was examined in 14 samples of colonal carcinoma and 16 samples of rectal carcinoma and paired normal tissues by SABC immunohistochemical staining and the results were analyzed by SPSS statistics software. Results: The positive particles of OPN were mainly distributed in the stroma cells of nomal tissues, and in the cytoplasm of tumor tissues. With the development of tumor, the positive particles of OPN increased gradually in the cytoplasm of tumor tissues, become less at the stroma .The express level of integrin β3 was medium in normal tissues, While it decreased significantly in tumor tissues. The express level of FAK and PI3K was significantly higher than that in normal tissues, and the positive particles were mainly distributed at the cytoplasm of tumor tissues. Conclusions: OPN may participate in the invasion and matastasis of medium-term and late-term coloretal carcinoma; Integrin β3 has negative relatively relationship with colorectal carcinoma:FAK and PI3K may participate in the invasion and metastasis of coloretal carcinoma.4.Effect of osteopontin on wound healing Objective: Wound healing is a complicated process.In general, wound healing is divided into three stages, inflammatory stage, proliferating stage and remodelling stage. During inflammatory stage, leucocyte,erythrocyte, platelet are recruited at sites of injury and release a series of cytokines and inflammatory medium. In proliferation stage, epithelial cells proliferate and migrate. Following proliferating stage, remodelling occur. Extracellular matrix proteins are markedly increased and redistributed. In recent years, we have found that osteopontin(OPN) can be released by smooth muscle cell in the process of endothelium damage. OPN can induce cell migration and adhesion. In addition, OPN recruits mononuclear cell. It plays an important role in the process of proliferation of vascular endothelium. These findings suggest that OPN might promote wound healing.The present study established wound healing model of rats to investigate the effect of OPN on wound healing. Methods: 1.Eighteen SD rats weighting about 300 g were used as experimental animals. The rats were randomly divided into OPN group, serum group, and EGF group. Dorsal part trauma model was established by removing full–thickness skin in a dorsal part. Then 1mg/mL OPN, 1mg/mL normal rat serum and EGF were applied to the wound. Specimens(granulation tissue)were taken at day 4 and 7 following treatment. The analysis of granulation tissue morphology was performed and type â… c ollagen content was detected 2.Immunohistochemistry was used to detect the typeâ… collagen. The paraffin sections were incubated with ployclonal antibody of anti-typeâ… collagen( 1 : 200 dilution ) ,positive particles appeared at extracellular matrix. 3.Western blotting of type â… collagen expression:The extracts from granulation tissue at different times after operation were separated on 8%SDS-PAGE, and then blotted onto nitrocellulose membrane. Themembrane was immunologically stained with anti-type â… collagen antibody. 4.The content of granulation tissue hydroxyproline: specimens were defated, dried and Hydrolysised for 24 hours, Chloraseptine oxidizing method was used to detect the content of granulation tissue hydroxyproline. Results: 1.The morphological changes: The general conditions of rats were good, and wound had no infection. At the first day after operation, the wound of serum group and EGF group was dry and the wound of OPN group had a little exudates. In the wound of all groups, granulation tissue appeared and epithelium proliferated at the 2th～3th day after operation. But granulation tissue of OPN group was less than that of serum group and EGF group. For 4～7 days after operation, granulation tissue of serum group and EGF group became mature, and had new capillary and fibroblasts. Granulation tissue of OPN was thin than that of serum group and EGF group. 2.The expression changes of typeâ… collagen in granulation tissue: The results of immunohistochemistry staining showed that positive particles appeared at extracellular matrix and the level of typeâ… collagen in granulation tissue of OPN group was less than that of serum group and EGF group. Western blotting suggested that the content of typeâ… collagen in granulation tissue of EGF group was 1.5 times as high as that of OPN group. The results of Western blotting were consistent with the results of immunohistochemistry. The results suggested that OPN almost had no effect on synthesis of type â… collagen and/or inhibited synthesis of type â… c ollagen. 3.The change of hydroxyproline contents in the granulation tissue. The data showed that the content of hydroxyproline had no significant difference among three group (P>0.05). Conclusion: 1.As an inflammatory chemotaxis factor, OPN played an important role that induced inflammatory reaction at early stage of wounding. 2.OPN might inhibit the proliferation of granulation tissue. |
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