| Background and Objectives:Since the1970s, the Percutaneous coronary intervention(PCI), which include the percutaneous transluminal coronary angioplasty(PTCA), in-stent planting and so on, has become the principal procedure for the treatment of coronary stenosis.However, the restenosis(RS) after PCI remains a serious problem for the failure of atherosclerotic therapy. Some researches indicated that the incidence of RS after PTCA or bare metal stents(BMS) was more than20-30%. Drug-eluting stents(DES) have been proven to reduce restenosis even more effectvely than PTCA or BMS(5-10%), although the problem was still not completely eradicated,espercially in the people with Diabetes mellitus. RS is a complex disease for which the causative mechanisms have not yet been fully identified. Our current understanding for the restenotic process is that an inflammatory response to the endothelial denudation and subintimal hemorrhage caused by balloon dilation and stent placement during PCI, resulting in the onset of several prolifetative processes, including vascular smooth muscle cell(VSMC) proliferation and migration, extracellular matrix formation and neointimal hyperplasia. Therefore, to prevent the RS after PCI, it is vital to learn the underling molecular and cellular mechanism of neointimal hyperplasia.Adenine nucleotide translo case (ANT), a vital mitochondrial protein located in the inner mitochondrial membrane, which is the major ingredient of mitochondrial permeability transition pore(MPTP). The ANT dimer exists in two conformations that are commonly referred to as the matrix state(m-state) and cytosolic state(c-state) which could control the mitochondrial permeability permeabilization(MMP) by facilitates the exchange of ADP and ATP across the inner mitochondrial membrane. Many researches indicated the ANT palys a key role in the control of cell death. For instance, in C.elegans, the ANT1homolog WAN-1promotes canonical apoptosis by cooperation with the core cell death machinery. Overexpression ANT1gene also could induce the cardiocytes and tumor cells apoptosis in vitro by trigger the way of mitochondrial mediated cell apoptosis.In our previous study, we have discovered that Overexpression ANT1gene also could induce the VSMCs apoptosis in vitro by upregulate the pro-apoptotic protein Bax. In order to identify whether overexpression ANT1gene can suppress the neointimal hyperplasia or not, we use the recombinant adenovirus vector which contains ANT1gene to transfect the rat carotid artery after balloon injury, then, detect the change of the carotid artery vessel by RT-PCR, Western-Blotting, Immunohistochemical Staining and TUNEL.Methods:1、The titer of the Ad-ANT1recombinant adenovirus vector is2×1011PFU/ml, it was recombined in our laboratory.2、72male Sprague-Dawley(SD) rats were randomly divided into4groups:a. sham-operated group(n=18), do not balloon injury the carotid artery; b. injured group(n=18), inject40ul PBS into the rat carotid artery after balloon injury; c. Ad-GFP group(n=18), inject40ul Ad-GFP recombinant adenovirus into the rat carotid artery after balloon injury d. Ad-ANT1group(n=18), inject40ul Ad-ANT1recombinant adenovirus into the rat carotid artery after balloon injury. Then, use the physiological saline to irrigate the vessel lumen30minutes later after the transfection.3、The left carotid artery were harvested at7days,14days and28days after transfection,0.5cm artery vessel was made into paraffin section to evaluate the neointimal hyperplasia of the carotid artery; the remained arteries were used to evaluate the expression of ANT1, Bax, Bcl-2by RT-PCR, Western-blotting, Immunohistochemical Staining.Results:1、The rat carotid artery balloon injury model were constructed successfully, and can be used in the subsequent experriment.2、The RT-PCR showed that the ANT1mRNA expression in the carotid artery of the Ad-ANT1group were significantly higher than the injured group and Ad-GFP group. It was indicated that the ANT1gene were tranfected into the rat carotid artery balloon injury model successfully.3、The Immunohistochemical Staining showed that the ratio of IA/MA of the Ad-ANT1group were significantly lower than other groups.4、The TUNEL assay showed that the apoptosis index of the Ad-ANT1group were significantly higher than other groups.5、The expression of the pro-apoptotic protein Bax of the Ad-ANT1group were significantly higher than other groups. But there were no differences of the expression of the anti-apoptotic protein Bcl-2among the three groups.Conclusions:1、The rat carotid artery balloon injury model was appropriate for the research about the mechanism of the neointimal hyperplasia or RS after injury.2、The recombinant adenovirus vectors which recombined in our laboratory could expression the ANT1gene into the rat carotid artery balloon injury model successfully.3、Overexpression ANT1gene suppress the neointimal hyperplasia by up-regulate the pro-apoptotic protein Bax which could induced the VSMCs apoptosis in the neointimal. |
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