The Biological Characteristics Of Co-cultured Endothelail Cells And Smooth Muscle Cells And The Effects Of 1Gs Static Magnetic Field On The Proliferation And Cytomembrane Ion Channel Of Co-cultured Endothelail Cells

[BACKGROUND AND AIMS]Rstenosis (RS) at dilatation site or in-stent position severely limited the long-term outcomes of percutaneous transluminal coronary angioplasty (PTCA) and stenting. Although many medical and mechanical methods of preventing RS had been developed, those were only proven unsuccessful. RS is the cascade reaction of various molecular and cell events in vessal wall, including two basic changes: blood vessal remodeling and neointimal proliferation. The former often took place in prime atherosclerosis, and the later mostly in the restenosis of the in-stenting site. The propose that the static magnetic fields (SMF) maybe prevented the RS firstly raised in our department and a great quantity of studies in vitro and in vivo had been done. The relative results indicated that different intensity of magneto-induction can exert paradoxical effents on the proliferation of human umbilical venous endothelial cells (HUVECs) and human umbilical arterial smooth muscle cells (HUASMCs). Once the intensity of the SMF increased to some extent, the damages would be caused in cells. Iatrogenic injury of endothelail cells (ECs) dysfunction of ECs and therefor inducing the proliferation and migration of vascular smooth muscle cells (VSMCs)to the neointimal hyperplasia. The recent studies suggested that cyclins cyclin-depandent kinases (CDKs) and CDK inhibitors (CDKIs) played an important role in vessal injurying and recovering process. This experiment aimed to investigate the biological characteristics of HUVECs and HUASMCs in the two-dimensional co-cultured pattern, which simulated the vessal enviroment in vivo, and the influence of 1 guass (Gs) SMF on their proliferation. In addition to, we focused on the cell cycle relative factors and electrophysiological properts of co-cultured HUVECs without or under IGs SMF.[METHODS]After the HUVECs and HUASMCs were succesfully obtained, cultured and passaged, we used the 2~4 passages of HUVECs and 5-8 passages of HUASMCs in this experiment. When two type of cells were put into the same plate, the culture medium changed into low serum culture for 48h(contain 5% fetal calf serum). The proliferative activity of co-cultured HUVECs and HUASMCs were inspected with the MTT, 3H-thymidine incorperation and flow cytometry (FLM) analyzing the cell cycle after propidium iodide (PI) and their growth status were obseraved under the light microscope. The angiotensin (Ang) II and L-arginine were then used to the single- and co-cultured plates at 24h after co-culturing to evaluate their action on the proliferative tendency of the two type cells. Immunohistochemistry and in-site hybridization were done to assess cell cycle relative factors and apoptosis in single- and co-cultured HUVECs with or without Ang II or L-arginine for 24h. Subsequently, we tested all the above items when the co-cultured cells pretreated by the IGs SMF for 48h. At last, the intracellular free calcium ([Ca2+]i) of single-cultured HUVECs and various co-cultured HUVECs were loaded with the fluoresent probe Fluo/AM and detected by Laser scanning confocal microscopy (LSCM), at same time the background non-selective cations current on their cytomembrane were recorded by the whole-cell patch clamp.[RESULTS]1. The proliferative activity of co-cultured HUVECs and HUASMCs was decreasing in the former and increasing in the later; the growth status of both cells went to resting phenotype, which were more similar to those in vivo.2. AngII and L-arginine had the proliferative tendency of single- and co-cultured HUASMCs not to change;3. The proliferative effect of L-arginine on the single- and co-cultured HUVECs revealed same tandency, but the inhibition of Angll on single-cultured HUVECs disapeared in co-cultured ones.4. The cell cycle relative factors participated in the HUVECs' cycle progression (in vitro) consisited of cyclinD and E CDK2 and 4 CDKI p27 and so on;5. when the co-cultured HUVECs and HUASMCs were exposed to the 1Gs SMF for 48h at same time, the results showed that the proliferation...