组织工程心脏瓣膜不同脱细胞方法的比较

Comparison of various acellular methods for tissue-engineered heart valves

背景:目前对几种脱细胞方法的效果各家观点不同。大都认为各种方法均可去除细胞,但实验结果却有较大差异。目的:对比胰蛋白酶法、十二烷基硫酸钠法和去氧胆酸钠法去除细胞后心脏瓣膜形态学及生物力学特征。设计、时间及地点:对比观察实验,于2007-10/2008-07在上海交通大学医学院细胞生物学实验室和上海交通大学医学院附属新华医院进行。材料:40个新鲜猪主动脉瓣120片瓣叶分为3组,分别为胰蛋白酶组、十二烷基硫酸钠组和去氧胆酸钠组,每组40片。方法:胰蛋白酶消化法:置于0.5g/L胰蛋白酶和0.2g/L乙二胺四乙酸,在37℃,体积分数为0.05CO2培养箱中振荡24h。十二烷基硫酸钠法:置于0.3g/L十二烷基硫酸钠和DNase,RNase中持续振荡24h。去氧胆酸钠法:首先在5g/LTriton-X100,5g/L去氧胆酸钠,0.2g/L乙二胺四乙酸中振荡24h,然后在2.5g/LTriton-X100,2.5g/L去氧胆酸钠和0.2g/L乙二胺四乙酸中破膜去污振荡48h,两阶段均加入DNase,RNase。主要观察指标:3种方法去细胞前后心脏瓣膜的生物力学特性变化。3种方法去除细胞程度和对胶原纤维、弹力纤维的影响。结果:去氧胆酸钠组拉伸率、最大拉伸应力、最大载荷均高于十二烷基硫酸钠组和胰蛋白酶组(P<0.01)。胰蛋白酶法脱细胞效果差,对基质破坏明显;十二烷基硫酸钠法可完全脱细胞,对基质破坏较大;去氧胆酸钠法完全脱细胞同时对基质破坏较少。结论:与胰蛋白酶法、十二烷基硫酸钠法比较,去氧胆酸钠法脱细胞效果好,能更好保护基质。

BACKGROUND： Presently, the acellular methods are various, but the experimental results are different. OBJECTIVE： To compare the differences of heart valve morphology and biomechanics after treated with either trypsin or sodium-dodecyl-sulfate （SDS） or Triton-X 100 and sodium-deoxycholate. DESIGN, TIME AND SETTING： A controlled experiment was performed at the Cytobiology Laboratory and Xinhua Hospital Affiliated to Medical College of Shanghai Jiaotong University from October 2007 to July 2008. MATERIALS： 120 fresh aortic valve leaflets of 40 pigs were equally divided into a trypsin group, SDS group and sodium-deoxycholate group. METHODS： Samples of the trypsin group were treated with 0.5 g/L trypsin and 0.2 g/L ethylenediamine tetraacetic acid （EDTA） under permanent shaking for 24 hours at 37 ℃ in a 0.02 volume fraction CO2 incubator. Samples in the SDS group were placed in SDS concentrations of 0.3 g/L with added RNase and DNase under permanent shaking for 24 hours. The left tissues were treated with 5 g/L sodium deoxycholate, 5 g/L Triton-X 100 and 0.2 g/L EDTA for 24 hours of shaking, and 2.5 g/L sodium deoxycholate, 2.5 g/L Triton-X 100 and 0.2 g/L EDTA separately for 48 hours of shaking, in addition to RNase and DNase. MAIN OUTCOME MEASURES： Valvular biomechanics characteristic changes of the before and after acellular treatment using three various methods. The acellular degree, and effects of the three methods on collagen fibers, elastic fibers. RESULTS： Extension rate, maximal tensile stress and maximal load were higher in the sodium-deoxycholate group than in the SDS group and sodium-deoxycholate group （P 〈 0.01）. The acellular outcome was poor using the trypsin method, and a significant destruction on the matrix could be found. The SDS method had good acellular outcomes, but also had a great destruction on the matrix. The sodium-deoxycholate method had good acellular outcomes, and also had little destruction on the matrix. CONCLUSION： Compared with trypsin and SDS methods, the sodium-deoxycholate method obtains an optimal acellular outcome and can protect the matrix.