Experimental Study Of The Effect Of Promoting Apoptosis Of Vascular Smooth Muscle Cell With Human Bcl-2 MRNA-cleaving Ribozyme

Percutaneous transluniinal coronary angioplasty (PTCA) is an effective method, which is widely used in the treatment of ischemic heart disease in recent 20 years. But 3 0-50% of restenosis following angioplasty hamper its use. So the research of restenosis has become the important parts of cardiology. The recent studies have shown that the lack of apoptosis is one of mechanisms which restenosis forms. It is a novel therapeutic strategy for rest enosis to promote vascular smooth muscle cells(VSMCs) apoptosis. Apoptosis is an active, manipulative and normal phenomenon, which is regulated by a series of gene. The gene therapy can affect apoptosis by charging the gene, which regulates apoptosis. Ribozymes are cataliytic RNA molecules that can cleave target RNA substrate based on sequence-specific recognition. The enzymatic specificity is mediated by variable flanked sequences complementary to the target RNA sequence surrounding the cleavage site. As ribozymes can cleave target RINA and can be reused in the procedure, they are taken as a more effective approach in gene therapy in comparison with antisense oligodeoxynucleotides. Bcl-2 family are anti-apoptotic molecules which can inhibit apoptosis caused by many kinds .5. of stimulates. The purpose of this study is to investigate the expression of Bcl-2 and rest enosis and to confirm that Bcl-2 mRNA-cleaving ribozyme has the effect of promoting apoptosis of human vascular smooth muscle cells (L{VSMCs), so as to provide the experimental data and academic foundations on the new genethrapy of restenosis.The main research methods and results are as follow: 1 .The immunohistochemisty(ABC) were used firstly to examine the specimens of coronary artery. We found that in the specimens of coronary artery in which the stent had been deposited there was high bcl-2 oncoprotein (77.5%,11/14),which in the normal coronary artery was less (25%,3112). The expression of bcl-2 in the specimens of coronary artery in which the stent had been deposited was high positive. This result shows that the high expression of bcl-2 may have relations with the inhibition of apoptosis after vascular injury and have played some role in restenosis. 2.The inducible bcl-2 mRNA-cleaving ribozyme eukaryotic expression vector (pMTr-neo) was constructed by inserting into pMTHSP7O-neo vector which contain the inducible Metallothionein-II(MT-II) promoter a cDNA fragment of bcl-2 ribozyme. The recombinant vector (pMTr-neo ) was proved correct by enzyme restriction pattern. 3.The HVSMCs was successfully cultured with a new method established by us. 4.The inducible bcl-2 mRNA-cleaving ribozyme eukaryotic expression vector (pMTr-neo) had been transfected into human VSMCs by lipofectin transfection .The DNA and RNA dot blot and mRNA situ hybridization was carried out, the results showed The expression and transcription of bcl-2 RZ was probed. The expression of bcl-2 protein in transfectants was .6. obviously down-regulated. 5.TUNEL analysis showed that 15.4% of the transfected HVMCs were positive. 6.Under electron microscope, the transfected HVMCs were clustered with increased nuclear density, the chromatin was c...