Transfection And Conditional Expression Of Angiotensin Ⅱ Type 2 Receptor Gene Mediated By Tetracycline-regulatable System And Its Effect On The Bio-Behaviour Of Rat Vascular Smooth Muscle Cells

Background: Arterial intimal thickening occurs in restenosis following angioplasty has generally been attributed to proliferation,migration of vascular smooth muscle cells (VSMCs) into the intima and vascular remodeling. Moreover, among a number of cardiovascular mediators, the renin-angiotensin system may be important particularly. At least two subtypes of angiotensin receptors have been identified: AT(1) and AT(2). The AT(1 )receptor mediates all of the known actions of Ang II in the cardiovascular system, such as vasoconstriction, increasing cardiac contractility and renal tubular sodium reabsorption, as well as vascular and cardiac hypertrophy. In contrast, less is known regarding the function of the AT(2) receptor. Evidence suggests that the AT(2) receptor inhibits cell proliferation and induces differentiation, apoptosis and regeneration. The AT(2) receptor has been shown to reverse AT(1) receptor-mediated hypertrophy, suggesting that these receptors exert opposing effects in the cardiovascular system. Although the physiological role of the AT2 receptor is still poorly defined, it may be implicated in inhibition of the proliferation and migration vascular smooth muscle cell (VSMC), which play a major role in the pathological changes of restenosis after angioplasty. The AT2 receptor is abundantly and widely expressed in fetal tissues, but present at low levels in adult tissues and re-expressed in certain pathological conditions such as vascular injury.We hypothesize that the low- or non-expression of AT2 receptor in adult and injury vasculature is the one reason of the emergency and development of restenosis after angioplasty. It may be beneficial to increase the level of AT2 receptor expression after angioplasty. Objective: The purpose of present study are 1) to construct the tetracycline-regulatable vector system to transfer angiotensin II (AngII) type 2 receptor (AT2R) gene into vascular smooth muscle cells(VSMC); 2) to establish the double stable cell lines of rat vascular smooth muscle cells(VSMC) to express AT2R; 3) to investigate the effect of AT2R on the accumulation of extracellular matrix and the regulation the cell cycle of VSMC in vitro.Methods: 1) The VSMCs, which got from the aorta of rat, were cultured by routinemethod. 2) recombinant tetracycline-regulated vector, pUHD10-3 containing rat AT2R gene was constructed by homologous recombination. 3)We established the regulatory system in two steps: at first, a stable VSMC cell line expressing rtTA had been constructed and characterized by transfected with pUHD 17-1hyg and selected by hygromycin B; in a second step, this line was used for transfer the gene of interest, AT2R gene, to VSMC to get the well establishing double stable VSMC lines. 4) The expression of AT2R and AT1R in VSMC were evaluated by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemistry respectively. 5) The effect of overexpression of AT2R on the mRNA and protein expression of Proiferating cell nuclear antigen(PCNA), Cyclin dependent kinase 2(CDK2), P21, P53, Matrix metalloprotease 2(MMP2), Osteopontin (OPN), Fibronectin(FN) were detected by RT-PCR, western blot and immunohistochemistry respectively. At the same time, the angiotensin II (AngII) type 1 receptor (AT1R) was also determined. And the effects of AT1R antagonist (CV-11974) and AT2R antagonist (PD123319) on aforementioned target were studied .Results: 1.The Doxcycline(Dox) - on gene expression system was constructed by recombinant DNA technique, including regulator plasmid pUHD 17-1hyg, response plasmid pUHD 10-3/AT2R, the luciferase-control vector pUHC13-3 and the selection plasmid pUHC13-3.2. Cultured rat aortic cells were idenfied as VSMCs by morphology and immunocytochemical stain.3. The expression of AT2R in double stable VSMC was increasing significantly after transferred by Doxcycline(Dox) - on gene expression system with addition of Doxcycline(1×10－3～104 ng/ml) , and the peak value detected by western blot was about 1×102ng/ml...