The Molecular Mechanism Research For Vascular Calcification Resistance Of Potassium

Objective: To investigate the effects of potassium on vascular smoothmuscle cell(VSMC) calcification and artery calcification in vitro and to revealthe molecular regulation mechanism of calcification by potassium.Methods:1. The separation and identification of the original generation of VSMCfrom mice. Separate and culture the original generation of VSMC fromC57BL/6J mice by the improved tissue pieces method, to observe the typicalcell morphology with optical microscope, identify the expression of VSMCspecific protein α-SMA by immunohistochemical method, detecte the α-SMAand SM22α gene expression of VSMC at mRNA level by real-timequantitative PCR.2. The calcification inhibition research of potassium on VSMC. Choosethe fifth generation of VSMC as experimental cells and divide the experimentinto three groups: there are normal control group (biological phosphorus),high phosphorus calcification group (2.6mmol/L Sodium phosphate) andpotassium intervention group (2.6mmol/L NaPi+1.0mmol/L KL). Culturethe cells for ten days and observe the cell growth situation by opticalmicroscope. Observe the cell calcification by Von Kossa staining. Detecte thegene expressions of α-SMA, SM22α, ALP, BMP-2, OPG and Runx2/Cbfα1inVSMC at mRNA level by real-time quantitative PCR. Observe the cellapoptosis by flow cytometry instrument. All the experiments repeat3times.3. The calcification inhibition research of potassium on mice aorta.Separate and culture the aorta from C57BL/6J mice, and divide the experiment into three groups: there are normal control group(biologicalphosphorus), high phosphorus calcification group(2.6mmol/L Sodiumphosphate) and potassium intervention group (2.6mmol/L NaPi+1.0mmol/LKL). Culture the mice aorta for twenty days and observe the vascularcalcification by Von Kossa staining.Results:1. Cell morphological identification. The cell morphology of miceVSMC was spindle and appeared the typical "valley-peak" growth state underthe optical microscope. It was showed by the-SMA antibodyimmunohistochemical research that a large number of brown granules can beseen in the wrapped slurry of VSMC. The RT-PCR analysis discovered thatthe expression of-SMA and SM22α in mRNA level were high.2. The microscopic observation of the cell. It can be seen under theoptical microscope that with the extension of culture time, the celltransparency in high phosphorus calcification group began to decline, theflocculent deposits were increased and the scopes were expanded gradually.On the other hand, compared with the high phosphorus calcification group,the flocculent deposits in potassium intervention group were decreased andscope narrow.3. The results analysis of VSMC by RT-PCR. Compared with the normalcontrol, the expressions of ALP-1、OPG/RANKL、BMP-2and Runx2/Cbf1at mRNA level in high phosphorus calcification group were obviouslyup-regulated(P﹤0.01). Compared with the the high phosphorus calcificationgroup, the expressions of ALP、 OPG、 BMP-2、 Runx2/Cbf1weresignificantly decreaed(P﹤0.01) and the expressions of-SMA、SM22were significantly increased(P﹤0.01) at mRNA level. 4. The cell apoptosis results.Compared with normal control group,theapoptosis rate of high phosphorus calcification group was increased(P﹤0.01);compared with high phosphorus calcification group, the apoptosis rate ofpotassium intervention group was decreased(P﹤0.01).5. The von Kossa staining results. The VSMC were dyed by von Kossastaining after being cultured for20days. From the von Kossa staining resultsit can be seen that, a numerous of dark brown calcium salt depositions in highphosphorus calcification group, a few of calcium salt depositions in potassiumintervention group, no obvious calcium salt depositions in normal controlgroup. From the above results we can say that the potassium ions have a storgeffect of calcification inhibition.Conclusion:1. The VSMC calcification and vascular calcification can be induced bythe use of high phosphorus culture solution.2. KCL could obviously suppress the VSMC calcification and vascularcalcification induced by the use of high phosphorus culture solution.3. Potassium chloride could inhibit the phenotype transformation ofVSMC and reduce the cell apoptosis, so as to prevent the occurrence ofVSMC calcification and vascular calcification.