Verification method for stimulating osteogenic differentiation of valvular interstitial cells by inorganic phosphate

Abstract

The invention relates to a verification method for stimulating osteogenic differentiation of valvular interstitial cells by inorganic phosphate. The method comprises the following steps: respectivelyperforming osteogenic differentiation culturing on extracted rat valvular interstitial cells by using CON, IP-OIM and OIM and then extracting protein to prepare a protein sample; after ALP staining and alizarin red staining on three kinds of protein samples, evaluating an osteogenic differentiation degree after staining by using ALP activity detection and a relative calcium content determination method, and detecting expression changes of BMP-2, RhoA and Rock-1 through a WB method and an RT-PCR method respectively; with the si-RNA technology, respectively interfering the expression of BMP-2 and RhoA and observing the relationship of mutual changes between the BMP-2 and the RhoA and whether the BMP-2 and the RhoA have the effect of resisting osteogenic differentiation or not after interference; and studying the influence of IP-OIM on SMAD1 / 5 / 9 phosphorylation as well as a mutual relation between two paths of BMP-2 / SMAD1 / 5 / 9 and RhoA / Rock-1. According to the invention, facts that calcification formation of valvular interstitial cells is caused by inorganic phosphate through BMP-2 / smald1 / 5 / 9 and RhoA / Rock-1 signal pathways and the BMP-2 / SMAD1 / 5 / 9 pathway is an upstream pathway of theRhoA / Rock-1 pathway in the valvular interstitial cell osteogenic differentiation process are determined; and a forward regulation effect is realized.

Description

technical field [0001] The invention relates to the technical field of osteogenic differentiation of valve interstitial cells, in particular to a method for verifying that inorganic phosphate stimulates the osteogenic differentiation of valve interstitial cells. Background technique [0002] Calcific aortic valve disease refers to the loss of elasticity of the valve leaflets due to valve degeneration or calcium salt deposition, resulting in varying degrees of left ventricular outflow tract stenosis. Valve interstitial cells (valvular interstitial cells, VICs) play a role similar to the cytoskeleton in the valve to maintain the shape of the valve leaflets. Under certain conditions, it can also differentiate into the direction of osteogenic phenotype. Valve interstitial cells are also considered as the main cells in the process of aortic valve calcification, and from the pathological characteristics of calcified valves, they have bone tissue-like features. [0003] In relate...

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Problems solved by technology

[0004] In the process of constructing the classic in vitro calcification model using classic organophosphates such as sodium β-glycerophosphate, there are long modeling time (14-21 days) and low efficiency Although some scholars have pointed out that high levels of inorganic phosphate in patients with chronic kidney disease can easily lead to calcification of blood vessel walls, the specific mechanism of osteogenic differentiation of valvular interstitial cells has not been studied in depth.

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