注射用胶原酶治疗腰椎间盘突出症体外作用机制

目的研究注射用胶原酶对磷脂酶A2活性的影响及对3种类型胶原蛋白的水解效率。方法不同剂量的胶原酶在不同时间内水解磷脂酶A2,其水解产物检测是通过SDS PAGE分析确认;以卵磷脂作为磷脂酶A2的底物,在反应体系中分别加入不同剂量的胶原酶以考察其对磷脂酶A2活性的影响;利用Lineweave r Burk双倒数法,求得磷脂酶A2水解卵磷脂的米氏常数(Km)以及反应体系含有胶原酶时的表观Km;分别以Ⅰ型、Ⅱ型和Ⅲ型胶原蛋白为底物,在100 min内,考察每个反应体系在间隔10 min时产物生成量,以比较注射用胶原酶对不同类型胶原蛋白的水解效率。结果注射用胶原酶不水解磷脂酶A2,但对磷脂酶A2活性具有抑制作用,且随胶原酶剂量的加大,抑制作用也随之增强,磷脂酶A2水解卵磷脂的Km为0.95 mol.L-1,含有胶原酶时的表观Km为3.76 mol.L-1;注射用胶原酶对3种类型胶原蛋白水解活性不同,其水解速率是Ⅲ型>Ⅱ型>Ⅰ型。结论胶原酶在体外对磷脂酶A2活性的抑制作用是竞争性;在3种类型胶原蛋白中,对Ⅲ型的水解作用最强。

Mechanism of collagenase for injection for treatment of herniated lumbar intervertebral disc in vitro

Objective To study the effect of collagenase injection(CI) on the activity of phospholipase A_2 and to compare the hydrolysis rates of three types of collagens.Methods The hydrolysis of phospholipase A_2 by three doses of CI in different times were examined.The hydrolytic products of phospholipase A_2 were analyzed by SDS-PAGE.Using lecithin as substrate of phospholipase A_2,the actions of phospholipase A_2 were tested by different doses of CI.The values of Michaelis constant(K_m) were quantified by Lineweaver-Burk's method for the different reactions.The production of each reaction was studied per 10 min within 100 min in order to compare the hydrolytic rates of three types of collagens.Results Collagenase did not hydrolyze phospholipase A_2,but could inhibit it.The degree of inhibition increased with the in crease of collagenase.The value of K_m is 0.95 mol·L~(-1) in the reaction of lecithin catalyzed by phospholipase A_2 and the apparent value of K_m is 3.76 mol·L~(-1) in the reaction containing CI.The three types of collagens had different hydrolysis rates.The order of hydrolysis rate is collage Ⅲ,Ⅱ,and Ⅰ.Conclusions The inhibition of CI on phospholipase A_2 is competitive in vitro.Collagenase has the highest hydrolysis rate for collage Ⅲ.