| Vascular smooth muscle cells (VSMS) are mainly present in the mold vessel, which constitute the basis of the blood vessel in the membrane material and perform certain physiological functions, regulation of vascular avoidance of diastolic and maintain the integrity of the vascular wall plays an important role. Recent studies have found, VSMCs proliferation and migration of vascular proliferative diseases is a core part of its molecular mechanism of pathology research in recent years, the proliferation of vascular disease and prevention hot and difficult. So grasping an efficient and simple method of vascular smooth muscle cells in culture is necessary. RNA interference is a gene found in recent years, a new blocking technology is a way to make the occurrence of a specific mRNA degradation, post-transcriptional gene silencing phenomenon. It has become an important research direction and focus of anti-tumor, anti-viral, regulation of gene expression, gene therapy, gene function studies and other fields. We know that the main function of unit L-Ca+ for al subunit, it is not only the voltage receptors, as well as ion-selective, while also binding sites, such as most of the calcium antagonist drugs. L-Ca2+ al subunit of rat aortic smooth muscle is mainly encoded by Cav1.2 and Cav1.3.This topic using tissue adherent primary cultured rat vascular smooth muscle cells, and then spread to 5-8 generations, with the construction of targeting Cav1.2, Cav1.3 gene siRNA vector transfected rat vascular smooth muscle cells, specific silence rat thoracic aortic smooth muscle cells Cav1.2, Cav1.3 gene expression. In addition, by MTT assay of siRNA vector transfected on vascular smooth muscle cell activity in rats. By using real-time quantitative RT-PCR and Western-Blot and other molecular biology techniques, we detect Cav1.2, Cav1.3 mRNA expression levels and protein expression levels to prove the inhibitory effect of this gene RNA interference level from biological macromolecules.The results showed that 24 hours after transfection, the transfected cells were positive after 48 hours, the fluorescence intensity was observed further enhanced. The transfection efficiency calculated was greater than 70%.72 hours after transfection, cells were collected, extracted mRNA was measured by PCR, the results can be seen Cav1.2, Cav1.3 mRNA expression level differences in the negative control group, the control group had no significant difference (P>0.05), the experimental group Cav1.2 and Cav1.3 mRNA expression was significantly lower than the control group decreased 86.54%, 88.38% respectively (P<0.01). After 72 hours transfection, cells were collected, extracted proteins were detected after Western-Blot observed bands visible, Cav1.2 blank control group and negative control group, Cav1.3 hybrid protein with brightness significantly stronger than the genome analysis results can be seen the negative control group, Cav1.2 blank control group, Cav1.3 protein levels showed no significant (P>0.05), the experimental group Cav1.2 and Cav1.3 protein than the control group decreased 80.36%,82.69% (P＜0.05).The results showed that transfection of siRNA expression vectors can inhibit vascular smooth muscle cells of rat Cav1.2, Cav1.3 expression of genomic expression Cav1.3 gene and protein expression at the transcriptional and translational levels, Cav1.3 of mRNA than negative control group and the control group was significantly inhibited. This method can provide an important scientific basis and reference for exploring the pathogenesis of vascular disease genes and so on. |
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