| [Objective] To investigate the relationship between bone marrow cell mobilization and phlebothrombosis, as well as to observe the effects of monocyte chemoattractant protein-3(MCP-3) on chemotactic function of macrophage in rats'venous thrombus mobilized by bone marrow cell so as to explore the effect and mechanism of bone marrow mobilization with the recombinant human granulocyte colony-stimulating factor (rhG-CSF) on venous thrombus organization and recanalization.[Methods] The adult male Sprague-Dawley rats were assigned to normal, sham-operated, thrombus and mobilization group. Normal group didn't experience any treatment. The same procedure was taken in sham-operated group and thrombus group, except a silk ligature was passed around the inferior vena cava but left untied. A daily dose of 25ug/kg rhG-CSF had been injected abdominal subcutaneously for 7 days since the operating day in mobilization group, while the same volume saline was injected in other groups.The peripheral blood was collected 30min preoperatively and 1, 3, 5, 7,10 and 14d postoperatively. Leukocyte count was detected by using an automatic blood cell counter, and the percentage of mononuclear cells in blood smear was counted under microscope. With application of ELISA, the level of MCP-3 in peripheral blood was detected. At post-operative 7th and 14th day, Inferior vena cava samples of thrombus group and mobilization group were collected, and laparotomy was performed to excise inferior vena cava which was under left renal vein and extanded to the confluence of left and right common iliac vein; thrombus orgnization rate and density of macrophages were observed by means of immunohistochemistry, image analyzer and electronmicroscope. Expression of MCP-3 mRNA in thrombus was analyzed via RT-PCR.[Results]1. Mononuclear cells count in peripheral blood Mononuclear cells increased significantly in mobilization group at 3rd, 5th and 7th day post-operatively (rhG-CSF: 2.92±0.36×109/L,3.28±0.26×109/L,4.48±0.33×109/L) compared with that of thrombus group (1.72±0.22×109/L,1.56±0.36×109/L,1.46±0.41×109/L) (P<0.05).2. Thrombus organization rate and ultramicroscopic observation Compared with thrombus group (14.64±2.32%, 63.21±8.68%), the organization rates in mobilization group at 7th and 14th day were 32.52±3.50%, 89.54±5.62% respectively, and the difference was significant (P﹤0.01). Ultramicroscopic observation suggested that, compared with thrombus group, there were a large number of macrophages which had active phagocytosis function in mobilization group with rhG-CSF treatment, new vessels and mature recanalization vessels were often observed around them.3. Macrophage densityMacrophage density (CD68 staining) of thrombus in mobilization group with rhG-CSF treatment (8.58±1.24%,P=0.0037) was significantly increased, compared with thrombus group (1.57±0.82%) at 7th day postoperatively; On 14th day after operation, macrophage density in mobilization group with rhG-CSF treatment(14.52±1.32%,P =0.0079) was also significanly increased compared with thrombus group (5.62±1.07%).4. Levels of MCP-3 of peripheal bloodMCP-3 level of peripheral blood were significantly increased at 7th day postoperatively in sham-operated group,thrombus group and mobilization group(96.53±8.21,99.81±7.96,138.44±10.01), compared with each group's level before operation respectively( P<0.05), and the difference was significant. Although at 14 th day postoperatively, the level of MCP-3 (108.8±9.26)decreased, it was still higher than peroperative level and also higher than that in sham-operated and thrombus group at 14 th day postoperatively(42.68±0.56,44.82±0.68, )( P<0.05), and the differences had statistical significance. 5. Expression of MCP-3 mRNAAs compared with thrombus group, levels of MCP-3 mRNA increased at 7th and 14th day postoperatively in mobilization group (P﹤0.01), the difference was significant; In thrombus group, expression of MCP-3mRNA at 14th day postoperatively decreased obviously than that at 7th day postoperatively, but there were no significant differences in mobiliztion group.[Conclusion]1. Mechanical injury of venous wall combined with stenosis is effective to stablely establish rat venous thrombosis model.2. rhG-CSF can increase chemokine MCP-3 in peripheral blood and expression of MCP-3mRNA in thrombus.3.The increase of MCP-3 in peripheral blood and the expression increase of MCP-3mRNA in thrombus play an important role in macrophage aggregation, suggesting that the chemotactic function of MCP-3 on macrophages is enhanced after mobilization by rhG-CSF(Macrophage homing to venous thrombus),therefore, the procedure of thrombus organization and recanalization is accelerated. |
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