IFNγ Inhibit Rat Vascular Smooth Muscle Cells Calcification Induced By High Phosphorus

Objective: The morbidity and mortality of chronic kidney disease(CKD) patients with cardiovascular disease(CVD) is significantly higher than the general population, the main reason is probably constitute the main risk factors for CVD and chronic kidney disease death more than 50% of the total mortality. More studies have shown that cardiovascular events, accounting for about 20% of coronary artery calcification. And different from traditional coronary calcification, CKD patients with vascular calcification occurs mainly in the film, which mainly composed of vascular smooth muscle cells. A large number of studies have shown that with the loss of renal function in patients with CKD,and the reduction of phosphorus clearance, then serum phosphorus liter high, this process promotes vascular calcification. Hyperphosphatemia is an independent risk factor for the promotion of vascular calcification in patients with CKD. A lot of basic research showed that hyperphosphatemia promote vascular smooth muscle cells phenotype to osteogenesis/chondroid combination by expressing the core factor of alpha 1(Cbfα1), upregulating BMP-2 and induce calcification.Transformation of VSMCs phenotype induced by high phosphorus is a key link in the process of vascular calcification, and also contains lots of other complicated factors, but the potential mechanism of vascular calcification has not been clearly known, the treatment and prevention of vascular calcification has yet to find effective means and methods. Research shows that interferon gamma plays a regulatory role in the osteoblast and osteoclast formation process, while the process of VSMCs phenotype transdifferentiation exactly similar to this.This study was to explore on the basis of high phosphorus induced vascular calcification increased interferon gamma can inhibit or reduce the degree of vascular calcification.Methods:1 Pimary culture to get vascular smooth muscle cells(VSMCs), and identify the cells by morphology and immune method, and then depict normal growth curve of smooth muscle cells.2 Experimental and Intervention groups: VSMCs were randomly divided into negative control group, the group of high phosphorus and IFN γintervention group, negative control group using low glucose DMEM medium containing 10% fetal bovine serum, high phosphorus group on the basis of normal medium added 10mmol/L β-glycerol phosphate for high phosphorus meduim. IFNγ intervention group were added IFNγchloride in the medium on the basis of high phosphorus, the final concentration of IFN γ were 100u/m L,the stimulating time is 3 days.3 Calcification assay: the calcification of VSMCs detected by using Alizarin Red stain of calcification and colorimetric method to determine the intracellular calcium content.4 Alkaline phosphatase(ALP) activity assay: take 3 to 5 cells seeded in 6-well plates, the density of cells is 2×105/pore, intervention 7 days, refused the medium, and then cells were washed two to three times with PBS, and added 500μl 0.1 % Triton X-100 to each hole at 4℃ environment, about 12-24 h, cells were lysed and then repeated pipetting into centrifuge tubes centrifuged by chemiluminescence detection ALP activity.5 By RT-PCR method to study effects of the different concentration of magnesium ions VSMCs Cbfα1,BMP-2,Smad1,Smad7 m RNA expression levels.6 Statistical methods: Statistical analysis of data using SPSS13.0 software for analysis. The experimental data were described by mean ± standard deviation( sx ±).Compared differences between two groups by t test, differences among multi-groups by one-way analysis of variance(ANOVA) and means of two subgroups comparison in multi-groups by Student-Newman-Keuls(SNK). With P<0.05 for the difference was statistically significant.Results:1 Primary culture of VSMCs: Use organization block adherent method to obtain the original generation of rat VSMCs, usually about 3-4 d a few cells can climb out from stick wall of artery tissue block, for long fusiform, scattered arrangement, the cell number increased significantly and confluent over time, about 6-7 d cells covered most of the bottle bottom, when the number of cells accounts for the 80-90% of the total bottom area can be represented. After represented cells were round or oval at first, stick a wall after can be gradually extend into spindle, abundant cytoplasm, nuclear lobes ovoid, crisscross overlap multiple cells growth, is a typical "peak to valley" performance. Detection of smooth muscle cells by immunocytochemistry method specific antibodies-alpha smooth muscle actin(α-SMA actin), that the cell cytoplasm strong expression is positive results, immune reaction products was brown.2 Alizarin Red calcification staining results showed: While compared with the negative control group, VSMCs calcification dyeing were increased in some extent degrees in the other groups, and IFNγintervention groups compared with high phosphorus group, the calcified nodules of VSMCs by Alizarin Red staining decreased.3 Calcium content test results showed: Compared with the negative control group, the levels of calcium content were significantly elevated in other groups, while compared with high phosphorus group, the levels of calcium content die down in IFNγ intervention groups, the comparative differences between groups with statistical significance(P < 0.05).4 ALP activity assay results: Compared with the negative control group, the level of ALP in VSMCs rised in other groups in some degree, while in contrast to the high phosphate and IFNγintervention groups.5 Results of RT-PCR to detect the expression of each group VSMCs in Cbfα1,BMP-2,Smad1,Smad7 m RNA display: Compared with the control group, the level of Cbfα1,BMP-2,Smad1 m RNA in VSMCs increased respectively in high phosphate and IFNγ intervention groups, while the Smad7 m RNA levels increased in the intervention group in contrast to the high phosphate group.Conclusion:1 This subject adopts thoracic aorta tissue block adherent method successfully obtained SD rat VSMCs, built a platform for VSMCs primary culture.2 High phosphate medium can induce VSMCs to calcify and with IFNγ, VSMCs calcification can be inhibited to some extent.3 The mechanism of IFNγ suppression the calcification of VSMCs induced by high phosphate may occur through inhibition of transdifferentiation and antagonizing calcium influx to achieve synergy.