The Role Of Connective Tissue Growth Factor In The Pathogenesy Of Scirrhous Gastric Carcinoma

Background and Objective:Scirrhous gastric carcinoma (SC), also called widespread and invasive gastric cancer or Borrmann typeⅣgastric cancer, has peculiar biological features. SC are characterized by diffuse invasive growth patterns with marked stromal fibrosis, showing the appearance of so-called"linitis plastica", and exhibit frequent peritoneal dissemination,lymph-node metastases and poor prognosis. The 5 years survival rate of scirrhous gastric carcinoma is only 5-20%. As a result, the pathogenesy of scirrhous gastric carcinoma became a hot issue in the fields of general surgery. At present, the pathogenesy of scirrhous gastric carcinoma has not been clarified completely, but more and more researches show that a large number of cytokines take part in the pathogenesy of scirrhous gastric carcinoma. It has been proved that scirrhous gastric carcinoma cells secrete plenty of cytokines, such as TGF-β, PDGF, IGF-Ⅱ, bFGF and etc. These cytokines promote the proliferation of interstitial fibroblasts and the production of collagen protein and metalloprotease, which lead to the interstitial fibrosis of scirrhous gastric carcinoma. On the other hand, interstitial fibroblasts secrete HGF,TGF-βand KGF, which enhance the proliferation and the invasive ability of scirrhous gastric carcinoma cells. These researches show that the unbalance of various cytokines plays a very important role in the pathogenesy of the interstitial fibrosis of scirrhous gastric carcinoma.Connective tissue growth factor (CTGF ) is a secreted growth factor with 38-kda molecular weight and belongs to the CCN family of immediate early genes (CCN2). CTGF has been shown to be widely existed in many human tissues and organs .It could be synthesized by a variety of cells including fibroblasts, vascular endothelial cells, vascular smooth muscle cells, epithelial cells, tumor cells andchondrocytes. Biological effects of CTGF include promotion of mitosis and proliferation, mediation of cell adhesion, stimulation of cell migration, promotion of formation and fibrosis of granulation tissue,development of cartilage and bone,angiogenesis and production of extracellular matrix (ECM). Many factors have been shown to induce CTGF expression, such as TGF-β, PDGF, EGF, FGF, VEGF. Hence, CTGF may be the common effector molecule of the fibrosis signal pathway downstream, and plays a pivotal role in the process of tissue and organ fibrosis. CTGF has been implicated in many normal physiological processes, including skeletal growth, limb regeneration, wound healing and angiogenesis. Conversely, CTGF overexpression has been linked with various pathologies. CTGF is highly overexpressed in numerous fibrotic diseases, including scleroderma, kidney fibrosis, hepatocirrhosis, lung fibrosis and atherosclerotic disease. The above researches give us a clue that CTGF has a very important application value in the diagnosis and treatment of fibrotic diseases, which became the most promising cytokine in the family of immediate early gene. At present, the function of CTGF in the pathogenesy of scirrhous gastric carcinoma has not been reported in the medicine literatures.The objective of this experiment is to investigate the role of CTGF in the pathogenesy of scirrhous gastric carcinoma from two aspects of clinical samples and cultured human cells in vitro; to study the expression of CTGF in gastric cancer tissue; to study the expression of CTGF in primary-cultured human gastric fibroblast; to study the influence of CTGF and anti-CTGF antibody on the proliferation, migration and ECM synthesis of human gastric fibroblast, all of which provide new thoughts for the clinical treatment of scirrhous gastric carcinoma.Methods:1. Immunohistochemical SP method was used to detect the expression of CTGF andα-SMA in 28 scirrhous gastric carcinoma (SC group),30 non-scirrhous gastric carcinoma(NSC group)and 30 para-cancer tissue samples (control group). Sirius -red staining was performed to assess the expression of typeⅠandⅢcollagen fibers in the above 3 groups. The correlations of the expression of CTGF,α-SMA and ECM were analyzed by statistics.2. Tissue explant was used to culture the human gastric fibroblast in vitro, then the primary cultured cells were purified, passaged and stored in liquidnitrogen. We observed the expression of vimentin and keratin in the cultured cell by immunocytochemical staining method in order to identify the origin of the cell. We survey the expression of CTGF in the cultured human gastric fibroblast by RT-PCR and immunocyte fluorescent staining to investigate the self-secretion of CTGF in human gastric fibroblast.3. (1) To observe the influences of different concentrations of CTGF (5, 20, 50, 100ng/ml) and anti-CTGF antibody (0.1, 0.5, 1ug/ml) on the proliferation of human gastric fibroblast by applying MTT colorimetry; (2) To survey the effects of different concentrations of CTGF and anti-CTGF antibody on the expression of collagenⅠmRNA of human gastric fibroblast by using RT-PCR technique; (3) To test the influences of different concentrations of CTGF and anti-CTGF antibody on the collagen protein secretion of human gastric fibroblast by using Hydroxyproline method; (4) To investigate the effects of different concentrations of CTGF and anti-CTGF antibody on the migration of human gastric fibroblast by using Transwell chamber method.Results:1. The results of immunohistochemistry staining show that CTGF is mainly expressed in the cytoplasm of gastric cancer cells, presenting granulated distribution. In the control group, CTGF is mainly expressed in the cytoplasm of gastric mucosa epithelial cells and presents weak expression. CTGF expression is not found in gastric gland cells and smooth muscle cells. The level of CTGF expression was significantly higher in SC group than that in NSC and control group (P <0.01). In comparison with control group, the expression of CTGF in NSC group was remarkably increased( P <0.01);α-SMA is mainly expressed in the cytoplasm of smooth muscle cells of gastric wall, vascular smooth muscle cells and myofibroblasts, presenting granulated distribution.α-SMA is not found in the cytoplasm of gastric cancer cells and the differences ofα-SMA expression between SC group and NSC group are not obvious (P>0.05). The results of Sirius-red staining show that the contents of typeⅠandⅢcollagen obviously increase in the order of control group, NSC group and SC group (P <0.01). The statistics results of experimental data demonstrate that the expression of CTGF had a positive relationship with that of typeⅠandⅢcollagen (P < 0.01). However, the expression ofα-SMA have no evident difference between SC and NSC group(P>0.05). There was no significant correlation between the expressionofα-SMA and that of CTGF,typeⅠandⅢcollagen (P >0.05).2. Human gastric fibroblast cultured by tissue explant grow well and have stable passage. We wiped off the mixed epithelioid cell with trypsin solution by repeated attachment method. The cultured cells present long shuttle or star shape, with long, narrow and transparent cell body. Cell nucleus is ovoid in shape, with abundant cytoplasm and clear cell membrane; Cells are arranged tightly without distinct boundary, which grow in the form of radiation or vortex shape. The results of immunocytochemical staining show that staining of vimentin is positive and the staining of keratin is negative, which are up to the cell-labeling features of fibroblast cultured in vitro. We find that primary cultured human gastric fibroblasts express CTGFmRNA and protein by RT-PCR and immunocyte fluorescent staining methods.3. 5～100ng/ml CTGF can promote the proliferation of human gastric fibroblast in a dose-dependent manner; 20～100ng/ml CTGF can up-regulate the expression of collagenⅠmRNA of human gastric fibroblast in a dose-dependent manner; 20～100ng/ml CTGF can stimulate the collagen synthesis of human gastric fibroblast in a dose-dependent manner; 20～100ng/ml CTGF can stimulate the migration of human gastric fibroblast in a dose-dependent manner. On the other hand, 0.1～1ug/ml anti-CTGF antibody can inhibit the proliferation of human gastric fibroblast in a dose-dependent manner; 0.5, 1ug/ml anti-CTGF antibody can down-regulate the expression of collagenⅠmRNA of human gastric fibroblast in a dose-dependent manner; 0.5, 1ug/ml anti-CTGF antibody can suppress the collagen protein synthesis of human gastric fibroblast in a dose-dependent manner; 0.5, 1ug/ml anti-CTGF antibody can restrain the migration of human gastric fibroblast in a dose-dependent manner. All the above experimental groups and corresponding contrast groups have obvious differences (P <0.05).Conclusion:1. The level of the expression of CTGF,typeⅠandⅢcollagen were significantly higher in SC group than those in NSC and control group. In comparison with control group, the expression of CTGF,typeⅠandⅢcollagen in NSC group were remarkably increased. The expression of CTGF had a positive relationship with that of typeⅠandⅢcollagen. The expression ofα-SMA had no evident difference between SC and NSC groups. There was no significantcorrelation between the expression ofα-SMA and that of CTGF,typeⅠandⅢcollagen. Hense, CTGF plays an important role in the pathogenesy of scirrhous gastric carcinoma characterized by stromal fibrosis. Myofibroblasts are not responsible for the desmoplastic reaction of scirrhous gastric carcinoma.2. Tissue explant is a convenient and reliable method to culture human gastric fibroblast in vitro. The primary-cultured human gastric fibroblast can be stably cultured in vitro, stored in liquid nitrogen and has stable passage, which provides sufficient and reliable cell models for the research on the pathogenesy of scirrhous gastric carcinoma. The results of RT-PCR and Immunocyte fluorescent staining prove that human gastric fibroblast itself secretes CTGF, which shows that CTGF takes into effect through the autocrine and paracrine manners. That provides experimental basis for further research on the relation between CTGF and human gastric fibroblast.3. Experimental results achieved by MTT, RT-PCR, Hydroxyproline and Transwell chamber methods show that within certain concentration range, CTGF can promote the proliferation, collagen synthesis and migration of human gastric fibroblast in a concentration-dependent manner, which shows that CTGF takes part in the process of the interstitial fibrosis of scirrhous gastric carcinoma and plays an important role in the process. On the other hand, within certain concentration range, anti-CTGF antibody can inhibit these effects in a concentration-dependent manner, which provides new ideas for the clinical treatment of scirrhous gastric carcinoma.In conclusion, CTGF is highly expressed in scirrhous gastric cancer cell; CTGF takes part in regulating human gastric fibroblast functional activities under the physiological and pathological conditions, and plays an important role in the pathogenesy of scirrhous gastric carcinoma.