注射用胶原酶对磷脂酶A_2活性抑制作用的体外实验研究

Studies on mechanism of collagenase solution for inhibition on activity of phospholipase A_2 in vitro

目的研究注射用胶原酶对炎性物质磷脂酶A2(PLA2)的水解作用和对其活性的抑制作用。方法制备不连续SDS-PAGE垂直平板凝胶,将3种酶促反应体系的不同酶解时间的反应液样品,进行电泳、固定、染色、脱色。同时在PLA2活性测定体系中,加入定量的胶原酶溶液,测定各反应体系的PLA2活性相当值。以不含胶原酶的PLA2活性测定体系为标准,求出不同活性单位胶原酶体系的PLA2相对活性,并依据实验所获得的数据,绘制Lineweaver-Burk图加以识别。通过对注射用胶原酶在PLA2水解卵磷脂过程中所起的作用,确定注射用胶原酶对PLA2活性的抑制机制。结果注射用胶原酶对PLA2无水解作用,但在体外对PLA2的活性有明显的抑制作用,其抑制类型为竞争性抑制,且抑制作用随胶原酶剂量的增加而增强。结论虽然注射用胶原酶在体外对炎性物质PLA2的活性有明显的抑制作用,但根据酶促反应动力学的基本理论,“针抵患处、酶达底物”是构成化学溶解术这一微创技术的基本要素和应用该技术时必须遵守的基本原则。

Objective：To investigate the hydrolysis of collagenase for injection to inflammatory substance-phospholipase A2 （ PLA2 ） and the inhibition to activity. Methods： Uncontinuous SDS-PAGE vertical slab gel were prepared. Reaction solution sample in different enzymolysis time of three kinds of enzymatic reaction system were electrophoresis, fixed, stained and decolored. At the same time, quantitative collagenase solution were added in PEA2 active measuring system and PEA2 active value of each reactive system were measured. As PEA2 active measuring system without containing collagenase standard to calculate PEA2 relative activity in different active unit collagenase system and draw Lineweaver-Burk diagram according to data from experiment. Acorrding to the action of collagenase for injection in the process of PLA2 hydrolyzing lecithin to ascertain the inhibitive mechanism of collagenase for injection to the activity of PEA2. Results： Collagenase for injection had no hydrolysis to PLA2 , but the inhibition was obvious in vitro. The inhibitive type was competitive inhibition and the inhibition increase as the increscence of collagenase dose. Conclusion：Although collagenase for injection has obvious inhibition to inflammatory substances-phospholipase A2 ＇s activity in vitro. The basic theory of enzy- matic reaction dynamics ＂needle to the site,enzyme to the substrate＂ constitutes the basic element of chemolysis of one kind of microtraumatic technique and the basic principle having to be obeyed as applying this technique.