| Peritoneal metastasis occurred in 40%-50% out of whole cases of advanced gastric carcinoma, which is a frequent cause of death in these patients. The formation of peritoneal metastasis of gastric cancer is a complex pathophysiological process, which is hypothesized to be related to cancer cell activity, peritoneal characteristics and other factors such as multiple adhesion molecules, proteolytic enzyme, cytokine, angiogenesis factor and so on.Chemokines belongs to one type of cytokines which have chemotactic effect. According to the relative position of cysteine, chemokines can be divided into four categories:C, CC, CXC, and CX3C. Chemokines and their receptors get involved in many physiological and pathological processes (inflammatory responses, lymphocyte homing, immune response, infections, autoimmune diseases, graft rejection and angiogenesis and so on) and play important roles in them. In recent years, more and more evidence emerged to demonstrate that chemokines are closely related to the tumor growth, invasion and metastasis. The role that chemokines play in the formation of the peritoneal milky spots metastasis of gastric cancer is still unclear. There were studies which had shown that Stromal cell-derived factor-1(SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) might participate in peritoneal metastasis of gastric cancer on the basis that AMD3100, the blocking agent of CXCR4, can inhibit peritoneal metastasis of gastric cancer. Omental milky spots is the lymphoid tissue in peritoneum and composed of macrophages and lymphocytes. Macrophages secrete macrophage-derived factors (MDC/CCL22, the CC chemokines subfamily members) when they are stimulated by external factors. The receptor of CCL22 is CC-type chemokine receptor 4 (CCR4). The purpose of this study is to assay the expression of chemokine receptor 4 (CCR4) in the primary gastric cancer and analyze the relationship between the expression of chemokine receptor 4 and the clinic pathologic parameters; investigate the relationship between chemokines and the formation of metastasis of gastric carcinoma; construct the mold of the mouse milky spot metastasis of gastric cancer and observe the structure of these metastasis; verify the hypothesis that the preference of metastasis of gastric carcinoma is the milky spot of peritoneal; inspect the expressions of MDC/CCL22 and their recptor CCR4 in the milky spot of peritoneal; explore the relationship between the MDC/CCL22-CCR4 axis and the milky spot metastasis of gastric carcinoma and provide new strategy to treat peritoneal metastasis of gastric carcinoma.Methods:Pathological specimens of 56 cases of gastric cancer,56 cases of lymph node metastasis,12 specimens of omental metastasis of gastric carcinoma,10 specimens of normal gastric mucosa were included. The expression of CCR4 in the all specimens were detected by immunohistochemistry. The relationship between the expression of CCR4 and clinic pathological parameter were analyzed by the single-variable analysis.The human gastric cancer cell lines MGC-803, AGS, BGC-823, MKN-45 and SGC-7901 and the mouse gastric cancer cell lines MFC were cultivated in RPMI 1640 or DMEM supplemented with 10% fetal bovine serum. The expressions of CCR4 at the level of mRNA and protein in cell line MGC-803, AGS, BGC-823, MKN-45, SGC-7901,MFC,were detected by RT-PCR and Western-blot respectively.CCL22 with different concentrations was supplemented to the culture medium of BGC-823 in which CCR4 was highly expressed. Subsequently, the effects of CCL22 on the proliferation and chemotaxis of CCR4 were assayed by MTT and Transwell plates.The expression of CCR4 at mRNA level were detected by RT-PCR in the MFC. The suspension of fluorescence labeling (Dil) MFC in which CCR4 was highly expressed were injected into the abdominal cavity. The omental were observed by confocal fluorescence microscope 12h later. The expression of MDC in milky spot were detected by immuno histochemistry.In addition,615 mice peritoneal cavity was injected 0.2ml MFC cell suspension (containing 5×106 cells in number) in order to product the peritoneal metastasis model of MFC. Include 10 mice which have bloody ascites 5 days later as the experimental group. Meanwhile, take the 10 mice which were injected normal saline in their abdominal cavity as the control group.Ascites were collected in two groups at 6th day,8th day,10th day and The concentration of CCL22 were detected by Enzyme linked immunosorbent assay.Results:1. CCR4 was expressed in 41 cases of primary gastric carcinoma and the positive rate is 73.21%. CCR4 was expressed in 36 cases of lymph node metastasis and the positive rate is 64.29%. CCR4 was expressed in 10 cases of omental metastasis and the positive rate is 83.33%. CCR4 was not detected in the normal gastric mucosa. The expression of CCR4 in the primary gastric carcinoma is highly related to the tumor differentiation (P< 0.05), however it is not related to the other pathological parameter such as age, tumor size, location, phage, stage and lymph node metastasis and so on.2. CCR4 was detected to be highly expressed in the 5 human gastric cell lines:MGC-803, AGS, BGC-823, MKN-45 and SGC-7901 by RT-PCR and Western-blot. MDC could promote the proliferation and chemotaxis of BGC-823 dose-dependently in a certain range of concentration.3. CCR4 was detected to be expressed in the cell line MFC. In the specimens of milky spot metastasis, CCR4 and CCL22 were detected by immunohistochemistry to be positive. The concentrations of CCL22 in the abdominal fluid in the group of MFC were higher than the control group, there are statistical significant difference between them (P<0.05).Conclusions:CCR4 was expressed in parts of gastric carcinoma. MDC/CCL22 could promote the proliferation and chemotaxis of BGC-823. The early omental metastasis of gastric carcinoma is prone to the milky spot. The axis of MDC/CCL22-CCR4 play a important role in the milky spot metastasis of gastric carcinoma and CCR4 maybe the new target for the treatment of milky spot metastasis of gastric carcinoma.
|
PDF Full Text Request