Vascular Cryopreservation Time In Rat And Its Impact On The Activity And Bax, Bcl-2 Gene Expression

Background:The incidence of congenital heart disease accounts for 6‰-‰of the survival rate for babies, and the complex congenital heart disease accounts for 0.5‰-2‰.A large number of complex congenital heart cases require biological or artificial materials, such as valved homograft conduit, valved xenograft conduit and other types of blood vessels with valves, to heal by rebuilding the right (left) ventricular outflow tract. Because of these characteristics-structural nature,central blood flow, anti-reflux, low immunogenicity, not easy to form thrombosis, and not easy attached by bacteria, etc., valved homograft conduit is applied in an increasingly wide range treatment of complex congenital heart disease. Compared with the artificial blood vessel and other types of blood vessels, the active valved vascular has the characteristics of stronger capacity of cell growth, low risk of long-term valvular dysfunction and calcification stenosis and long life of blood vessels, so saving the activity of valved homograft conduit is the experimental research direction. The key point of saving the activity of valved homograft conduit lies in the acquisition, processing, cooling, preservation and minimizing the valved vascular cell necrosis and apoptosis in the rewarming process. As Bax genes to promote cell apoptosis and inhibit the apoptosis of Bcl-2 gene, the previous studies have confirmed that Bax gene expression can contribute to the ischemia-reperfusion injury model of myocardial cell apoptosis; while in the study of induction of rat myocardium apoptosis, the expression of Bcl-2 gene decreased the apoptosis of cardiac myocytes. In the past, the detection of vascular activity was carried out through the detection of glucose metabolism rate. Such kinds of methods detect the vascular activity of cellular metabolism without adopt the method of detecting the gene and protein activity. Therefore, base on gene and protein detection, we explore the vascular activity after frozen at different times.Objective:To study the activities and the expressions of Bax and Bcl-2 gene of rats'valved conduit by cryopreserve different times,and to probe whether genetic detection will become one of the index of valved conduit activition.Methods:80 wistar rats were divided into four groups:20 cases of fresh blood vessels (A group),20 cases of frozen vessels for 3 months (B group),20 cases of frozen vessels for 6 months (C group), and 20 cases of frozen blood vessels for 9 months (D group). By application the method of glucose metabolism to detect the Bax, Bcl-2 protein expression in immunohistochemical assay samples of metabolism of blood vessels, streptavidin peroxidase, SP.Results:1. There are no significant differences between the results in each group of glucose metabolism (P> 0.05).2. Microscopic examination of blood vessels in each group show that there are three-tier structures of the valved conduit and flexibility. In group A and group B, vascular endothelial cell has shown such characteristics:integrity layer, close connection, not obvious shedding, clear nucleus, tightly packed fiber layer. Meanwhile, Group C and group D have such characteristics:partial layered cortex, partial cells loss, disclose cell connection and tightly packed fiber layer.3. Positive rate of Bax protein in blood vessels is increasing with the extension of cryopreservation time. Protein positive rate of Bcl-2 has nothing to do with the length of cryopreservation time.Conclusion:1.There was no significant difference in the glucose metabolism results of cryopreserved valved blood vessels in rats and fresh glucose metabolism in rats (P<0.05), prompting that there is valved vascular activity in each group.2.There are significant difference in the Bax gene expression of cryopreserved valved vascular with the time extension, suggesting that with the extension of freezing time, cell apoptosis will be increased, and Bax gene can be regarded as one of the indicators for detecting valved vessel activity.3. There is no difference in the positive expression level of each group in BCL-2 gene, suggesting that the expression or over expression in BCL-2 gene may be one of the reasons for the cells can not tolerate the frozen preservation.