| Calcific aortic valve disease is a slowly progressive disorder, which can be divided into aortic sclerosis (ASc) and calcific aortic stenosis (CAS). In the developed countries, CAS is the most frequentheart valve diseases. CAS is no longer considered as a passive degenerative disease, but instead as an active process triggered by pro-inflammatory cues. For the limitation of valve donors from CAS patients and the long duration of animal models, it would be imperative to establish in vitro models.The aortic valve interstitial cells (AVICs) play an important role in the development of CAS. Most of the in vitro studies on CAS were accomplished with pig AVICs, and were involved in different calcified inducer, which making the comparability of conclusion between different studies poor. Although SD rat being one of the most common laboratory animals, it rarely be used in CAS studies, partly for the difficulty of isolation and culture of SD rat AVICs.The aims of our study were to isolate and culture SD rat AVICs. then to investigated the calcified ability of AVICs with extended culture time without the calcified inducer and detect the mechanisms in the calcification of AVICs, meanwhile investigated the effect of atorvastatin and amlodipine on the calcification of AVICs. Part1The culture of SD rats aortic valve interstitial cellsObjective:To establish the culture of SD rats AVICs.Methods:Culture SD rats AVICs in explant method, and make the observation of primary and passaged cells. Detect the a-SMA and vimentin expression by IF staining and investigate ultrastructure characteristics by TEM and SEM. Results:1AVICs were migrated out of the explants within2～3days and displayed a mixed of elongated or irregular morphologies; reached70～80%confluence7-8days later and passaged routinely.2AVICs were positive for a-SMA and vimentin.3AVICs attached to the microscope slides tightly, with extensive and irregular-shape by SEM.4There were abundant organelles in the AVICs, such as prominent rough endoplasmic reticulum and plentiful myofilaments by TEM.Conclusion:1Establish the culture of SD AVICs in explant method.2AVICs were myofibroblasts. Part2The establishment of cell model of spontaneous calcification of aortic interstitial cell in SD ratsObjective:To observe that AVICs calcified spontaneously whether or not.Methods:Culture the AVICs in explant method, and detect the calcifi-cation in Von Kossa, alizarin red S stain and a calcium assay kit at different day.Results:1AVICs aggregated and formed small cell nodule after4～6day culture, and the number of nodules become more and the diameter bigger at day12than that at day6.2The cell nodules were positive for Von Kossa and alizarin red S stain, which indicated that they were calcified nodules. The statistical analysis of the alizarin red S indicated that there were statistical significance between different time groups (F=106.167,P<0.001). the content of alizarin red S at day12were higher than that at day0and day6.3The statistic analysis of calcium indicated that there were statistical significance between different time groups (F=116.379, P<0.001), the content of calcium at day12were higher than that at day0and day6. AVICs calcified spontaneously with extend culture time.Part3The mechanism of spontaneous calcification in aortic interstitial cell in SD ratObjective:To detect the expression of osteogenic gene in the formation of calcified cell nodules.Methods:Culture the AVICs in explant method, detect the expression of osteogenic factors, such as a-SMA/Cbfa1/OC/ALP/BMP-2/BMP-4, with RT-PCR and western-blot analysis and the activity of ALP with a ALP assay kit at different day.Results:1The statistic analysis of the ALP activity indicated that there was statistic difference between different time groups(χ2=139.396, P<0.001), the ALP activity was lowest at day12, higher at day0and highest at day6.2The statistic analysis of the osteogenic gene expression (Cbfal/OC/BMP-4) indicated that there was statistic difference between different time groups (F=410.1G6,χ2=7.200, F=38.463, P<0.005), the Cbfal/OC/BMP-4mRNA were lowest at day0. higher at day6and highest at day12.3The statistic analysis of the ALP gene expression indicated that there was statistic difference between different time groups (χ2=7.200, P=0.027), the ALP mRNA were lowest at day0, higher at day12and highest at day6.4The statistic analysis of the BMP-2gene expression indicated that there was statistic difference between different time groups (F=56.565, P<0.001), the BMP-2mRNA were lowest at day0, higher at day6and day12.5The statistic analysis of the α-SMA gene expression indicated that there was statistic difference between different time groups (F=89.886,P<0.001), theα-SMA mRNA were lowest at day12, higher at day6and highest at day0.6The statistic analysis of the ALP protein expression indicated that there was statistic difference between different time groups (χ2=7.261, P=0.027), the ALP protein expression were lowest at day0, higher at day12and highest at day6.7The statistic analysis of the BMP-2/4protein expression indicated that there was statistic difference between different time groups (χ2=7.200, F=452.482, P<0.05), the BMP-2/4protein expression were lowest at day0, higher at day6and highest at day12.Conclusion; The differentiation of AVICs into osteoblasts-like cells may play an important role in the calcification process. Part4The effects of Atorvastatin and Amlodipine on the calcification of aortic interstitial cell in SD ratObjective:To analysis the effect of Atorvastatin and Amlodipine on the calcification of aortic interstitial cell in SD rat.Methods:Culture the AVICs in explant method, and detect the calcium and ALP activity at different day in different medicine.Results:1The statistic analysis of the calcium indicated that there was statistic difference between different time groups (F=116.379, P<0.001) under the controlled group factor, the calcium was lowest at day0and day6, higher at day12.2The statistic analysis of the calcium indicated that there was statistic difference between different medicine groups (F=3.655, P=0.023) under the controlled time factor, the calcium in10-6mol/L Aml group was higher than that in10-6mol/L Ato group.3The statistic analysis of the ALP activity indicated that there was statistic difference between different time groups (F=139.396, P<0.001) under the controlled group factor, the ALP activity was lowest at day12, higher at day0and highest at day6.4The statistic analysis of the ALP activity indicated that there was no statistic difference between different medicine groups (F=0.515, P=0.675) under the controlled time factor.Conclusion:There was no significant effect of Atorvastatin or Amlodipine on the calcification of AVICs. |
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