Studies Of Sever Factors' Effects On The Bovine Artery Smooth Muscle Cell Calcification And MRNA Differential Expressions In The Calcifying Process

Background: Vascular calcification is common phenomenon, which occurs more frequently in aging process and various kinds of disease such as coronary artery disease, hypertension, diabetes millets and uremia, causes most of the death and disability. It is almost invariably associated with atherosclerotic (AS) plaque formation. Resent studies have suggested that plaque calcification is an active process similar to osteogenesis. However details of mechanism by which vascular calcification be induced remains unclear, and made it of the most complicated mechanisms in the body. Some factors that can increase the mineralization of bone also play a role in the calcification of blood vessel cells. Here we want to demonstrate that 1,25(OH)₂D₃, TGF-βand IGF-1 which often found in the AS plaque can also affect the calcification of blood vessel just as they did in the bone cells.Among the many mechanisms related to vascular calcification,molecular mechanisms remains the most attractive one. Several important findings have suggested that genes responsible to osteo-specefic-non-collagenous-proteins were of most characteristics,such as osteopontin, osteocalcin, BMP (bong morphogenic protein), but they may probably present in the calcilied areas other than blood vessels.To further investigate the mechanism directly related to blood vessel, we used the calcified VSMC to screen the differentially expressed cellular mRNA for further study.Objectives: (1). Investigate the effect of 1,25(OH)₂D₃, TGF-β and IGF-1on the process of BASMCs calcification; (2). Screen the differentially expressed mRNA of BASMCs in the calcifying process.Methods: Based on the vascular calcification models in vitro, bovine aorta smooth muscle cell was cultured in DMEM contain 10mmol/L β-glycerophophate to induce calcify. 1,25(OH)₂D₃, TGF-β and IGF-1with differnt concentrations was added tD the calcnying and Standardmedium. The calcium deposition and cellular ALP was assayed by abiochemical method; Osteocalcin in cultUre supernatant was quanunedby radioimmunoassay. For the molecular research, we used thedifferential disPlay method tO ideneq the differential expressed mRNAfor both tWo cells, used reverse Nowhern Blot and Nothern Blot tOcondrm the differential expression cDNAs. The bioinfOrmatics sourcewas utilized tO get more infOrmation of these cDNAs.Rfsults: 5 -GP induced extensive calcium deposition of BASMCsafter 10 days of culture. Calctum deposition in the cellular layer wasincreased 24 times compared tO the standard medium and the calciumdeposition was confirmed by von Kossa stain. 1O9Inol/L vitamin D3 inthe medium increased the calcium deposition of calcified cultUred celi by16.25% affer 10 days of culture (P>0.05) but without statistic sighficance.whereas no change was deteCted in the st8ndard medium. During thecalcifying formation cellular ALP was gready increased with the tOpincrease in the 8th day with 201.76% more than the control. In the 10thday, there was a slight decine but was also 53.90% more than calctfiedcontrol. The ostEocalcin concentration in the cultUre supernatant wasincreased by 58.3%. Increased the VitD3 concentration in calcifiedmedium to 1pe and 1fr7 mol/L, the calcium dePosition of the cells wasunexpectedly decreased 22.32% and 35.89% respectively (P<0.05).Using Transforming Growth FactOr- fl as another factOr to study thecalcified process, we found that 1O'g/L TGF D didn't change thecalcium deposition of calcilied BASMCs as well as the standard culturemedium cells. Cellular ALP increased in a manner similar tO the VitD3treatinent, with the great6st increase in the 8th day, but the ost6ocalcinCDncentfation had no change. Increasing the TGF-b concentrahon ofcultUre medium to 1pe and 1O'g/L, hags remained the same,suggestEd that TGF- 0 have no ehat in the calcium deposition ofBASMCs although increased the ALP activity of those cells.Insulin-like growth factO...