一种体外流动钙化模型的建立

目的构建一种基于模拟生理流体环境、且可便捷、高效反映心血管植入物钙化特性的体外测试模型,为心血管植入物的快速开发提供新的手段。方法选择新鲜牦牛牛心包(青海裕泰畜产品有限公司,中国);选择3周龄Wistar雄性幼鼠5只,体质量(50±5)g。采用常规戊二醛(GA)与处理牛心包交联方法,制备GA交联的牛心包材料(Yak-GA)。在大鼠皮下埋植14、28、56 d做体内钙化实验。将橡胶管、瓣叶钙化舱、体液模拟液溶液瓶、蠕动泵组成体外流动钙化模拟装置,将Yak-GA置于瓣叶钙化舱,于不同流速(0、2、10 mL/min)下1.5倍模拟体液(SBF)转流14 d,测定钙离子浓度、pH值变化。利用SPSS软件对体内外钙化数据进行拟合,通过拟合优度分析,筛选出最佳的流速并获得体内钙化预估方程。结果Yak-GA体内植入14、28、56 d,组织钙沉积量分别可达到(52.17±11.01)μg/mg、(104.40±21.34)μg/mg、(171.06±15.11)μg/mg。体外钙化模拟实验中,对比0、2、10 mL/min流速下、相同时间点溶液钙损失,2 mL/min流速溶液钙损失量高,材料钙化进展最快;14 d时2 mL/min流速溶液钙损失量与0 mL/min流速比,差异有显著统计学意义[(2.07±0.07)mmol/L vs(1.14±0.10)mmol/L](t=13.41,P<0.01);与10 mL/min流速比,差异也有统计学意义[(2.07±0.07)mmol/L vs(1.89±0.01)mmol/L](t=3.55,P<0.05)。2 mL/min流速体内外钙化数据拟合优度R^(2)=0.941,所得的评估方程为:当1.65<C浓度≤2.05 mmol/L时,公式为Y=-126.71X+407.74;当2.05<C_(浓度)≤3.75 mmol/L时,公式为Y=e^((7.46–1.22X))(X=钙化液中钙离子浓度;Y=体内模型组织中钙沉积量);模型残差在(2.36±11.36)μg/mg之间波动。另外,研究中发现pH值变化与钙离子浓度变化线性相关性可达到0.94,具有极强相关性,相关性方程为Y=2.63×pH+0.37。结论成功开发了一种简单、有效、可实时监测钙化过程的体外流动钙化模型,其中2 m L/min为该系统最佳流速,该流速下所构建的模型高效且具有良好的体内外相关性。

Changes of chymase,angiotensin converting enzyme and angiotensin Ⅱ type 1 receptor expressions in the hamster heart during the development of heart failure

Background Little is known about the role of dual angiotensin Ⅱ forming pathways during heart failure. In the present study, the changes of chymase and angiotensin converting enzyme （ACE） expressions in the failing hearts of hamsters were analysed. Methods Heart failure was induced by ligation of left anterior descending branch of the coronary artery. Chymase, ACE and angiotensin Ⅱ type 1 receptor （AT1R） mRNA levels were analysed by reverse transcription polymerase chain reaction （RT-PCR）. The activities of chymase and ACE were determined by radioimmunoassay （RIA）. Myocardial collagen fibre analysis was performed under optical microscope. Results Left ventricular systolic pressure （LVSP） and maximum left ventricular developed pressure increase rate （ dp/dtmax, mmHg/s） gradually moved lower at 2, 3,4 and 8 weeks after operation. On the other hand, left ventricular end-diastolic pressure （LVEDP） increased gradually after operation. Compared with the control group （3.55±0. 06, 4.79±0.70）, the heart weight/body weight ratio in operation group had increased significantly at 4 weeks and 8 weeks （4. 28±0. 43, 6. 17±0.73） （P 〈0. 01 ）. Collagen staining showed that the quantity of myocardial collagen fibre increased significantly in the operation group. RT-PCR showed that the chymase mRNA level in the operation group was consistently greater than that in the control group. ATIR mRNA level was also increased significantly at 3 weeks and 4 weeks, both being 1.3 times that of the control group （ P〈0.01 ）, whereas ACE mRNA level was not changed. Higher activity of chymase was detected in operation group, being 4, 8, 13 and 19 times that of the control group at 2, 3, 4 and 8 weeks （P〈0.01 ）, respectively. ACE activity was also significantly higher at the same time, being 7, 10, 10 and 3.5 times that of the control（P〈0.01）. Angiotensin Ⅱ （Ang Ⅱ） level in operation group increased significantly, being 2.5, 2.7, 3.5 and 2 times that of the control group at 2, 3, 4 and 8 weeks, respectively （P 〈 0. 01 ）. Conclusions A dual Ang Ⅱ forming pathway from both ACE and chymase in the hamster hearts plays an important role during the development of heart failure. At the decompensatory stage, the reduction of AngⅡ level may be associated with the decrease of ACE activity.