| Purpose: Establish a stable restenosis model of rat and investigate expression of the Apolipoprotein J/Clusterin(Apo J/CLU) in the restenosis vascular, then study the relationship between the Apo J/CLU and restenosis and the potential mechanism.Method: 40 wistar rats were divided equally into experimental group and control group. After injected heparin via venae subclavia, 2F Fogarty catheter were inserted into the carotid artery of experimental group rat to injure the artery. The control group received sham-operation. The rats were collected venous blood and killed at 1,2,3,4 weeks after operation, separated the carotid arteries and removed 0.3cm of the right carotid artery to embed into 10% neutral paraffin for pathology examination; the remaining arteries were placed into liquid nitrogen and stored in the-80℃ fridge for biochemistry experiment. Morphology change of the carotid artery was observed weekly through microscope after H&E staining, then detected the expression of the Apo J/CLU protein in the carotid artery by immunohistochemistry, detected the Apo J level in the serum by enzyme linked immunosorbent assay(ELISA), at last detected the expression of the Apo J/CLU m RNA in the tissue by Real-time PCR.Result: ①There were 17 rats survived in the experiment group, survival rate is 85%. Intimal hyperplasia were observed in all the survival rats’ right carotid artery, the animal model is successful. Means of operation time is 34.19±6.09 minutes. ②HE staining: Carotid intima of rats appeared significant hyperplasia after injury. With the passage of time, the degree of thickening became more and more serious. A large number of vascular smooth muscle cell(VSMC) migrated to the intimal, with strong activity of migration and proliferation. In 4 weeks after operation, the activity of the VSMC in the wall side began to decline. Measurement of the intima / media area ratio(internal/medial, I/M) represented the degree of intimal thickening, we found that the I/M of control group close to 0. Every week after operation in experimental group, the I/M was larger than that of control group. The I/M of the fourth week was the most highest, and there was significant statistical difference compared with the previous 2 weeks. ③Immunohistochemistry: Apo J/CLU can be detected in carotid artery of the experimental group at each time point. It expressed in the VSMC’s cytoplasm oftunicae media vasorum or neointimal, the expression of Apo J/CLU protein was the most strongest in 3rd week, and declined in 4th week and all the control group failed to detect the Apo J/CLU protein. ④ ELISA: There was no difference in Apo J level before and after operation neither in control group or experimental group. ⑤Real-time PCR: There was no difference between experimental group and control group in the expression of Apo J/CLU m RNA in carotid artery in 1st week. It was higher in experimental group than control group in 2nd week, and was highest in 3rd week, and declined in 4th week.Conclusion: The modeling method is practical and repeatable. It has a high survival rate and also successful rate. VSMC’s migration and proliferation induced intima hyperplasia in carotid after balloon injury. The migration and proliferation activity in neoimtia were strong, but it decline in 4th week. The expression of Apo J m RNA and protein in VSMC of injured vascular increased significantly. They were highest in 3rd week, and decline in 4th week. It suggests that Apo J may be closely related to vascular injury and intimal hyperplasia after injury. The high expression of Apo J in vascular was mainly derived from the VSMC synthesis of injured tissues, but not from system. The amount of Apo J in the local synthesis was not enough to cause the changes of Apo J in serum. We assume that Apo J may be a kinds of compensatory which response to vascular injury, and it may plays a protective role in restenosis through inhibiting the activity of proliferation and migration on VSMC. |
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