The Study Of Th17/Treg Cells On The Effect And Mechanism Of Aortic Valve Calcification

Objective:To study whether Th17/Treg cells taking part in the development of aortic valve calcification and the mechanism to promote aortic valve calcification progression.Methods:First, aortic valves were divided into two groups(n=10):the control group,aortic valves came from patients being subjected to heart transplantation because of dilated cardiomyopathy, excluding valves with pathological change; the calcification group,aortic valves came from patients being subjected to valve replacement because of isolated aortic valve calcification. Exclude patients with rheumatism and endocarditis medical record. Every sample was tested by von kossa stain or immunohistochemistry analysis and western blot or reverse transcription polymerase chain reaction(RT-PCR). Second, using aortic valve interstitial cell culture and induction of differentiation of Th17cell technology in vitro, human aortic valve interstitial cells were cultured and passage three to five times, randomly divided into five groups(n=10):the control group, adding lml RPMI-1640; the experiment group1, adding lml culture supernatant of Th17cells; the experiment group2, adding lml culture supernatant of Th17cells and IL-17antibody (50ng/ml); the experiment group3, adding lml culture supernatant of Th17cells and IL-17antibody (50ng/ml) and receptor activator of nuclear factor-κB ligand (RANKL)(10ng/ml); the experiment group4, adding lml culture supernatant of Th17cells and RANKL antibody(10ng/ml). After48hours, using cell alizarin red stain to detect the degree of valve interstitial cells calcification, western blot and RT-PCR to detect the level of alkali phosphatase and bone morphogenetic protein-2expression.Results:First, Th17/Treg cells were not present in the control group, but they simultaneously emerged in the calcification group. In the calcification group, the number of Th17cells infiltration was higher than Treg cells (19.70±2.67VS10.70±1.89, P=<0.01). Second, in five groups, calcium nodus can be found. However, in the experiment group1and the experiment group3, the number of calcium nodus was higher than other three groups. Respectively, comparing with the control group, the experiment group2and the experiment group4, the level of ALP and BMP-2protein expression was higher in the experiment group1(0.743±0.053VS0.180±0.026, P=<0.01;0.901±0.059VS0.396±0.032, P=<0.01),(0.743±0.053VS0.249±0.030, P=<0.01;0.901±0.059VS0.421±0.031, P=<0.01),(0.743±0.053VS0.250±0.026, P=<0.01;0.901±0.059VS0.424±0.037, P=<0.01). Comparing with the experiment group3, the level of ALP and BMP-2protein expression was less in the experiment group2(0.249±0.030VS0.717±0.063, P<0.01,0.421±0.031VS0.880±0.052, P<0.01).Conclusion:Our study, for the first time, demonstrates:Th17/Treg cells take part in the development of aortic valve calcification and the degree of Th17cells infiltration is higher than Treg cells. On the one hand, Th17cells can stimulate the progression of chronic inflammation in aortic valve tissue by secreting inflammatory factor IL-17; on the other hand, IL-17can stimulates aortic valve interstitial cells transformation into osteoblast-like cells through RANKL/RANK signal pathway. By regulating the tendency of Thl7/Treg cell differentiation maybe interfere in the progression of aortic valve calcification.