Shi Yi, Zhou Ye-bo, Cai Yan, Teng Xu, Song Jun-qiu, Duan Xiao-hui, Zhang Wei-zhen, Qi Yong-fen
Department of physiology and pathophysiology, Peking University School of Basic Medical Sciences, Beijing 100191, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2010 Apr 18;42(2):131-6.
To explore the effect of age on vascular calcification induced by vitamin D3 and nicotine.
Vascular calcification in rats was induced by administration of vitamin D3 plus nicotine (VDN treatment). After six weeks, Von Kossa staining, calcium content, alkaline phosphatase activity, phosphorus and calcium content in plasma were assayed. Carotid blood pressure, cardiac function and the relative amounts of osteopontin (OPN), osteoprotegerin (OPG), matrix Gla protein (MGP), bone morphogenetic protein-2 (BMP2) mRNA level and smooth muscle actin-alpha (alpha-SMA)protein level were measured.
Compared with control group, the systolic blood pressure(SBP)of the rats of 2,8 and 16 months with vascular calcification respectively increased by 20.7%, 29.4% and 22.2% (P<0.05); the left ventricular systolic pressure (LVSP) respectively increased by 13.6%, 21.1% and 16.2% (P<0.05); + LVdP/dtmax respectively increased by 49.1% (P<0.01), 21.4% and 13.1% (P<0.05); -LVdP/dtmax respectively increased by 56.3% (P<0.01), 24.4% and 11.3% (P<0.05). Aortic calcium contents of the 2-, 8- and 16-month calcified rats were respectively 2.62-fold (P<0.05), 24.87-fold (P<0.01) and 10.01-fold (P<0.05) of the age-matched control group. As compared with the aortic calcium contents of calcified groups at different ages, the calcification group of 8 months had higher aortic calcium content than those of 2 and 16 months, which were respectively, 5.28-fold and 2.63-fold (P<0.05). Compared with the control groups, alkaline phosphatases activity (ALP) of calcification groups increased respectively by 126.6%, 115.2% and 227.9% (P<0.01) in the 2-, 8- and 16-month rats. As compared with the ALP activity of calcified groups at different ages, ALP activity of aortic calcification group of the 8-month-old rats was higher than that of the 2-month-old and 16-month-old rats, which increased by 176% and 75% respectively (all P<0.01). Von kossa staining for calcification showed positive staining as black/brown areas within the main, large, nodular structures as shown in extracellular matrix and cytoplasma in VDN groups at different ages, especially in the 8-month-old VDN group, with the most dispersed calcific nodules deposited and a few of the elastic fibers of the medial layer collapse. The mRNA expressions of OPN, OPG, MGP, BMP2 were up-regulated (P<0.01 or P<0.05) and protein levels of alpha-SMA were down-regulated in different calcification groups(P<0.05). The mRNA levels of OPN in 8-month-old calcification group increased by 3.41-fold (P<0.01) and 1.34-fold (P<0.05) respectively compared with the 2-month-old and 16-month-old calcification groups. And the alpha-SMA protein expression levels were lower at calcification groups in different ages, which were respectively equivalent to 17.6% of the 2-month-old control group (P<0.01), 11% of the 8-month-old control group (P<0.05) and 41.7% of 16-month-old control group (P<0.01).
SD rats of 2, 8 and 16 months can all be used to duplicate vascular calcification model induced by vitamin D3 plus nicotine and the 8-month-old rat has the most sensitivity to the calcification treatment, which means that the 8-month-old rat may be the most appropriate age for the study of vascular calcification.
探讨年龄对维生素D3和尼古丁诱导的血管钙化的影响。
通过给予维生素D3加尼古丁（VDN处理）诱导大鼠血管钙化。六周后，进行冯科萨染色、测定钙含量、碱性磷酸酶活性、血浆磷和钙含量。测量颈动脉血压、心脏功能以及骨桥蛋白（OPN）、骨保护素（OPG）、基质Gla蛋白（MGP）、骨形态发生蛋白-2（BMP2）mRNA水平和平滑肌肌动蛋白-α（α-SMA）蛋白水平的相对含量。
与对照组相比，2个月、8个月和16个月血管钙化大鼠的收缩压（SBP）分别升高20.7%、29.4%和22.2%（P<0.05）；左心室收缩压（LVSP）分别升高13.6%、21.1%和16.2%（P<0.05）；+LVdP/dtmax分别升高49.1%（P<0.01）、21.4%和13.1%（P<0.05）；-LVdP/dtmax分别升高56.3%（P<0.01）、24.4%和11.3%（P<0.05）。2个月、8个月和16个月钙化大鼠的主动脉钙含量分别是同年龄对照组的2.62倍（P<0.05）、24.87倍（P<0.01）和10.01倍（P<0.05）。与不同年龄钙化组的主动脉钙含量相比，8个月钙化组的主动脉钙含量高于2个月和16个月钙化组，分别为5.28倍和2.63倍（P<0.05）。与对照组相比，2个月、8个月和16个月大鼠钙化组的碱性磷酸酶活性（ALP）分别升高126.6%、115.2%和227.9%（P<0.01）。与不同年龄钙化组的ALP活性相比，8月龄大鼠主动脉钙化组的ALP活性高于2月龄和16月龄大鼠，分别升高176%和75%（均P<0.01）。不同年龄VDN组的钙化冯科萨染色显示，在细胞外基质和细胞质中的主要大结节结构内有黑色/棕色区域的阳性染色，尤其是8个月龄VDN组，钙化结节沉积最分散，中层的一些弹性纤维塌陷。不同钙化组中OPN、OPG、MGP、BMP2的mRNA表达上调（P<0.01或P<0.05），α-SMA蛋白水平下调（P<0.05）。8个月龄钙化组的OPN mRNA水平与2个月龄和16个月龄钙化组相比分别升高3.41倍（P<0.01）和1.34倍（P<0.05）。不同年龄钙化组的α-SMA蛋白表达水平较低，分别相当于2个月龄对照组的17.6%（P<0.01）、8个月龄对照组的11%（P<0.05）和16个月龄对照组的41.7%（P<0.01）。
2个月、8个月和16个月的SD大鼠均可用于复制维生素D3加尼古丁诱导的血管钙化模型，8个月龄大鼠对钙化处理最敏感，这意味着8个月龄大鼠可能是研究血管钙化最合适的年龄。
