优化明胶酶谱法检测自发性高血压大鼠血浆MMP-2和MMP-9活性

目的以优化明胶酶谱法检测自发性高血压大鼠(SHR)血浆基质金属蛋白酶-2(MMP-2)与-9(MMP-9)的活性。方法以含明胶的SDS-聚丙烯酰胺凝胶电泳酶谱法为基础,改变孵育时间、工作液成份,采用不同pH值的孵育液及不同冻融次数的样品,观察对基质金属蛋白酶(MMPs)活性的影响。并以优化的酶谱法检测Wistar及自发性高血压大鼠血浆MMPs的相对活性。结果凝胶孵育时间由42 h缩短为17 h不影响酶谱法检测结果。省略漂洗步骤及洗脱液仅使用2.5%Triton X-100、孵育液中去除NaN3及NaCl且电泳后步骤将去离子水更换为蒸馏水也不影响实验结果。孵育液pH值在7.2～8.8范围内均可用于酶谱法。血浆样品反复冻融多达6次不影响酶谱法检测结果。用优化的酶谱法检测结果显示,与Wistar大鼠相比,SHR大鼠血浆MMP-2与MMP-9活性显著增高。结论优化酶谱法与常规方法相比更为简便经济,检测大鼠血浆MMP-2与MMP-9活性所得结果一致。

Modified gelatin zymography for detecting MMP-2 and -9 activity in plasma of spontaneously hypertensive rats

Objective To improve the method of zymography for MMP-2 and MMP-9 detection in plasma of spontaneously hypertensive rats (SHR). Methods The effects of changed incubation time and working solution components, different pH values of developing buffer and plasma freeze / thaw cycles on the activity of MMPs were observed based on gelatin-containing SDS-polyacrylamide gel electrophoresis (SDS-PAGE)-dependent zymography. The relative activity of plasma MMPs was assayed by optimized zymography in Wistar and SHR rats. Results Gel incubation time reduced from 42 h to 17 h did not affect the test results of zymography. Rinsing step was omitted and renaturing buffer only containing 2.5% Triton X-100, NaN3 and NaCl were removed from developing buffer and deionized water was replaced for distilled water in steps after electrophoresis did not affect the test results of zymography. The pH value within 7.2~8.8 of the developing buffer can be used for zymography. Repeated freezing and thawing plasma samples up to 6 times did not affect the test results of zymography. The activities of MMP-2 and MMP-9 detected by optimized zymography were significantly increased in SHR compared with Wistar rats. Conclusions The optimized zymography is simpler and more economical and the experiment results are the same compared with conventional method.