同种异体肌腱羟脯氨酸测定的正交试验

An orthogonal test for spectrophotometry of hydroxyproline in allogeneic tendon

摘要:

[目的] 探讨不同因素对检测同种异体肌腱中羟脯氨酸 （hydroxyproline, HYP） 含量的影响。[方法] 使用分光光度计，基于正交试验的方法，对同种异体肌腱进行 HYP 含量检测，以样品的酶解时间（4、12、24 h）、酶解的水浴温度（37℃、 60℃、100℃），根据样品在酶解过程中需要盐酸优化酶解，设定酸酶比 （1∶2，1∶1，2∶1），样品酶解时的水浴震荡频率 （180 r/min、200 r/min、250 r/min） 4 个单因素变量交替试验，得到最优化检测因素。[结果] 单因素分析结果表明，在其他变量不变的情况下，酶解时间 12 h，水浴温度为 60℃，酸酶比为 2∶1，保证 pH 值为 7.4 左右，200 r/min 震荡频率，能保证 HYP 酶解效果最佳，正交试验及极差分析表明酸酶比影响作用最大，其他影响因素依次为水浴温度、震荡频率和时间。[结论]同种异体肌腱 HYP 含量检测，需调整 pH 值、温度、震荡频率及酶解时间保证酶的活性做高，使样本检测率达到最高，以期合理使用资源，同时达到最佳的检测效果。

Abstract:

[Objective] To investigate the influence of different factors on the spectrophotometry of hydroxyproline (HYP) in allogeneic tendon. [Methods] The HYP content of allograft tendons was detected by spectrophotometer based on orthogonal test. Four factors, including enzymolysis time (4 h, 12 h, 24 h) , water bath temperature (37℃, 60℃, 100℃), acid-enzyme ratio (1∶2, 1∶1, 1∶1), and water bath oscillation frequency (180 r/min, 200 r/min, 250 r/min) were set to explore the optimal assay conditions. [Results] The results of single factor analysis showed that under the condition that other variables remained unchanged, the enzymolysis time was 12 h, the water bath temperature was 60℃, the acid-enzyme ratio was 2∶1, the pH value was about 7.4, and the shock frequency of 200 r/min could ensure the best enzymolysis effect of HYP. Orthogonal test and range analysis showed that the acid-enzyme ratio had the greatest effect. Other influencing factors are bath temperature, oscillation frequency and time. [Conclusion] In spectrophotometry of HYP content in allograft tendon, pH value, temperature, shock frequency and enzymolysis time should be adjusted to ensure high enzyme activity, so as to achieve the highest detection rate of samples, so as to rationally use resources and achieve the best assay efficiency.