| BackgroundCoronary heart disease (coronary heart disease, CHD) is a serious threat to the health of the modern people, causes a heavy social burden of public health. According to statistics released by the World Health Organization in the year of2006, in the15-60year-olds around the world cause of death, stroke and coronary heart diseases were ranked at the fifth and the second. According to the data released by the Ministry of health of China, in the year of2005, Chinese urban residents’mortality of coronary heart disease was46.3/100000. The causes of coronary heart disease are still not fully understood, studies show that coronary heart disease is caused by multiple risk factors, including dyslipidemia, male, age, hypertension, diabetes, obesity, reduced physical activity, smoking, family history. Coronary heart disease symptoms often appear at people over40years old,80%of patients who underwent nonfatal myocardial infarction were over65years old. The treatments of coronary heart disease includ drug therapy, percutaneous coronary artery interventional therapy (Percutaneous coronary intervention, PCI) and coronary artery bypass grafting (Coronary artery bypass grafting, CABG). In the late1970s, PCI was first applied in clinic, in1984, China successfully carried out the first PTCA operation, after decades of development, from the initial simple balloon dilatation to the later bare metal stent (Bare mental stent, BMS) implantation, till now, the drug eluting stent (Drug elunting stent, DES) is widely used, as the advance of technology and the new equipment, PCI has become one of the relatively mature means for the treatment of coronary heart disease.Multicenter randomized clinical trials show that:coronary drug eluting stent can significantly reduce neointimal hyperplasia and restenosis rate (in5years of about9%～10%), alleviate the symptoms, improve the quality of life. Drug eluting stents (DES) is widely used while significantly reducing restenosis after PCI occurs, however, drug eluting stents, such as rapamycin eluting stents, in the inhibition of vascular smooth muscle cell proliferation has also led to stent implantation endothelial repair delay, easy to cause the thrombus relative incidences. At the same time, all the patients who underwent DES implantation need to receive dual antiplatelet therapy for at least12months, which increases the risk of postoperative hemorrhage.MTOR is a mammal animal cell expression of a serine/threonine protein kinase. Studies show that, mTOR pathway activation is involved in the formation and development of atherosclerosis, and it havs synergy effect with inflammation, lipid metabolism disorders. The abnormal regulation of mTOR signaling pathway is closely related to the occurrence and development of atherosclerosis, the traditional mTOR inhibitors mainly rapamycin and its derivatives, could inhibit the production of monocyte/macrophage related cytokine, including MCPland IL-6. Suzuki found that rapamycin can effectively reduce the MCP-1mRNA level. Basso found that the application of rapamycin inhibits of the atherosclerotic process and the effect is independent of cholesterol lowering therapy. Intravascular ultrasound imaging (IVUS) is a new kind of imaging techniques developed in recent years in coronary interventional operation, its value in interventional therapy has become increasingly significant, because it can accurately display the properties of atherosclerotic plaque and the artery wall, be able to accurately judge the stent apposition or in-stent restenosis, thus it has become the one of the most important assistant means for the diagnosis and treatment of coronary artery disease, we have known that the oral administration of rapamycin have playd a positive role on plaque, we hope that with the intravascular ultrasound technology, to assess the effect that oral administration of rapamycin combined with bare metal stents for the treatment of restenosis. At the same time, for the histological and pathological aspects, we used immunohistochemical techniques, transmission electron microscopy, to detect the target vascular tissues of a-actin, mTOR, collagen, lipid and other indicators, in order to seek BMS implantation plus oral rapamycin can have a similar effect with the DES implantation, if this method is feasible, then a new scheme can be addicted to percutaneous coronary artery interventional therapy, in the prevention of stent intrinsic stenosis at the same time, also can reduce the in-stent thrombosis incidence, and reduce the time for postoperative dual antiplatelet treatment, and reduce the incidence of late stent thrombosis.Objective(1) To establish AS animal model; (2) Use intravascular ultrasound to test the abdominal aorta atherosclerosis plague pattern of the experimental animal and implant a stent in a specific location;(3) To analyze the blood biochemical indexes before and after intervention;(4) the application of intravascular ultrasound (IVUS), pathological and immunohistochemical techniques, to find out oral rapamycin inhibits in-stent restenosis and its molecular mechanism;(5) To find out that whether bare metal stent implantation combined with oral rapamycin can achieve a similar effect with rapamycin-eluting stent implantation, for clinical prevention and treatment of coronary heart disease and to provide theoretical and experimental evidence.Materials and methods24male New Zealand rabbits in purebred, adaptive feeding for1week, each rabbit by the ear margin vein,4ml blood specimen were got before and after the adaptive feeding, to analyze TC, LDL, L, HDL-L etc changes. Balloon injury abdominal aorta of all the experimental rabbits,3%sodium pentobarbital30mg/kg anesthetized the experimental animals, puncture the right femoral artery, located the balloon catheter of diameter3. Omm, length15mm into the descending aorta for about20cm,14big air atm filled the balloon, than repeated pull back and forth3times to cause abdominal aortic endomentrial intimal injury, then ligature the femoral artery at the far end, suture the wound. Intramuscular injection of antibiotics was given to each rabbit to prevent infection. Give a high fat diet (1%cholesterol, each120-140g/Day) to each experimental rabbit for8weeks.8weeks end, each rabbit was exsanguinated4ml venous blood from ear margin veins to analyze lipid index, and experimental animals were randomly divided into3groups, each group of8rabbits:bare stent group (group BMS), bare stent+oral rapamycin group (group BMS+RAPA), drug-eluting stent group (DES group). All animal were3%sodium pentobarbital anesthesiaed, exposed the right femoral artery of each rabbit, made abdominal aortic angiography on each rabbit, to observe the positions of the abdominal aortic plaques, then apply IVUS examination, the specific operation as follows:separation of the right femoral artery,under assisted of the guide wire and the expansion tube,100u/kg of heparin was given intravenous anticoagulation, with the help of the guide wire and the expansion pipe, the intravascular ultrasound detector was sent to the narrow part of the vessel, then slowly pull back the IVUS detector, to measure each index of the site where the stent is to be implantated such as:minimal luminal diameter (MLD), external elastic membrane area(EEMA), lumen area (LA), plaque area (PA), plaque burden (PB) lesions and reference sites of external elastic membrane area (EEMA), lumen area (LA), plaque area (PA), plaque thickness diameter (Dmax) and its contralateral plaque thinnest diameter (Drain). According to the formula of eccentric plaque index (EI) and vascular remodeling index (RI):EI=Dmax/Drain, EEMA/RI=lesions reference site proximal and distal EEMA average value. All stent experimental groups animals were selected the appropriate location (position is chosen below the renal artery lmm), with16～20atm to release the stent, stent types were3.0*12mm, then use IVUS to measure index, retained image, save the imaging video, then ligature the femoral artery at the far end, suture the wound, give each rabbit intramuscular injection, antibiotics to prevent infection for two days, and2500u/d heparin for one week. After the operation, all the experimental rabbits continue to give a diet of1%cholesterol for4weeks. In BMS+RAPA group of rabbits, give rapamycin orally for4weeks, the doses of0.5mg/kg/d.4weeks end, after exsanguinating each animal4ml venous blood to analyze lipid levels, use IVUS to measur those indexes and calculated vessel lumen lost of stent groups. After that, all animals were killed, leaving bracket part and between the proximal and distal vascular tissue. Then put all the experimental animals to death, keep whole stent section vessel as well as proximal and distal it. First we get lmm plaque part vessel to analyze macrophagus by Transmission electron microscope, the reat samples were transected into two same parts. One part was kept in formaldehyde for HE staining, Immunohistochemical staining, Masson staining etc, and the other part was kept in-80℃for molecular biology study and oil red staining. Statistical analysis using SPSS10.0statistical software for windows. All values are expressed as mean±SD, count data is expressed as a percentage, multiple group comparison using ANOVA procedure, a value of P<0.05for the difference was statistically significant.Result(1) The IVUS results show that PA, PB of BMS+RAPA and DES groups are significantly less than that of group BMS (P<0.01),while MLD of BMS+RAPA and DES groups is significantly higher than that of group BMS. Lumen loss4weeks after the implantation stent was significantly less than that of group BMS (P<0.05);(2) After building the model of rabbit, blood lipid indexes significantly increased (P<0.05)(Table1),4weeks after the intervention, the differences among the three groups were not statistically significant (Table2)(3) Pathological test of group BMS shows that intimal hyperplasia after stent implantation significantly, lumen area stenosis is heavier than that in the BMS+RAPA group and DES group (P<0.05). BMS+RAPA group of vascular endothelium is better, while group DES endothelial repair delayed obviously, the stent is directly exposed in the lumen.(4)4weeks after the intervention, level of mTOR expression in BMS+RAPA group and DES group plaques was significantly lower than that in BMS group (P<0.05).(5)4weeks after the intervention, electron microscopic test shows that group BMS has a obvious aggregation of foam cells in plaques, aggregation of foam cells in BMS+RAPA group and DES group are significantly less than BMS groupConclusion(1) Oral administration of rapamycin can inhibit intimal hyperplasia where stent is implanted, reduce in-stent restenosis, oral administration of rapamycin combined with bare stents treatment has a similar effect that reduce lumen area loss, reduce plaque burden as the rapamycin eluting stent treatment. Compared with bare stents group, can effectively reduce the plaque area and lumen area loss, lower the incidence of restenosis.(2) Oral administration of rapamycin combined with bare stent and drug-eluting stent have a similar effect that inhibits in-stent restenosis.(3) By inhibiting localized on the mTOR, Rapamycin reducs local plaque inflammation and lipid deposition, reduces proliferation of stent smooth muscle cell, and prevent the in-stent restenosis.(4) Oral administration of rapamycin can effectively reduce plaque progression. Bare metal stent implantation combined with oral administration of rapamycin, have obvious advantages in the prevention of restenosis and reduce complications such as bleeding. So it has a good prospect of clinical application. |
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