Establishing A Mouse Strain Of Specific Deletion Of Smad4 In Smooth Muscle Cells

The TGF-βfamily belongs to a superfamily known as the transforming growth factor-βsuperfamily which has diverse functions in most cells. It comprises of TGF-β1, TGF-β2 and TGF-β3. TGF-βsignals through receptor serine/threonine kinases and intracellular Smads proteins. Smad4 is an important factor of TGF-P signaling which can form complexes with the phosphorylated R-Smads and enter the nucleus, where they bind to DNA and interact with transcription factors to regulate gene expression. Many recent studies have demonstrated that the TGF-P signal pathway has an important role in Cadiovascular diseases. The technology of gene knock out is a new method to delete targeted gene in vivo. The aim of this study was to establish a smooth muscle cell specific Smad4 knockout mouse and to analysis its primary phenotype, intending to obtain a model of Cadiovascular disease to further study the effect of TGF-P in the development of the disease. Also the present study aims to establish a primary culture method of mouse vascular smooth muscle cell to obtain primary vascular smooth muscle cell from Smad4 gene knockout mice to further study the functions of TGF-P in vascular smooth muscle cell in vitro.A mouse specifically expresses Cre reconbinase in smooth muscle cells was selected to mate with ROSA26 reporter strain to get a SMA-Cre; ROSA26 double transgenic mice. LacZ staining in the SMA-Cre; ROSA26 double transgenic mice demonstrated that the Cre reconbinase specifically expressed in the vascular smooth muscle cell within the vessel wall. Then the SMA-Cre mice was mated with a mouse strain that carries Smad4 conditional alleles (Smad4co/co) to obtain the smooth muscle cell specific Smad4 knockout mice (SMA-Cre; Smad4 flox/flox). The results manifested that Smad4 was indeed deleted in the vessel wall of the SMA-Cre; Smad4flox/flox mice. The deletion of Smad4 in smooth muscle cell did not result in embryonic lethality, approximately 70% of the SMA-Cre; Smad4flox/flox mice dead between 6 to 8 weeks of age. The mutant mice exhibited abdominal aortic aneurysm in 6 weeks of age. Reformed enzymatic technique was established in the present study to obtain primary smooth muscle cell culture which was characterized by immunofluorescenc. Aftardward, the primary smooth muscle cells was isolated from the SMA-Cre; Smad4 flox/flox mice,Western blot and Real-time PCR were used to detect the expression of Smad4. It demonstrated that the isolated cells were stable heredity with Smad4 deletion.The results of the present experiments manifested that a smooth muscle cell specific Smad4 knockout mice was established which could be used as a perfect model of abdominal aortic aneurysm to study its pathogenic mechanism and to find therapy method for it. Besides, a primary culture method of smooth muscle cell was established which was used to isolate primary smooth muscle cells form the Smad4 mutant mice to further study the impact of TGF-P in smooth muscle cells in vitro.