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目的:从人胎盘中分离、纯化组织金属蛋白酶抑制因子3(TIMP3),并对其对基质金属蛋白酶1(MMP1)的抑制作用进行评价。方法:利用含4mol/L尿素的TrisHCl缓冲液(pH8.0),从除去多数水溶性蛋白的胎盘匀浆液中提取难溶性蛋白。经CM52阳离子交换树脂和SephacrylS200凝胶过滤两步柱层析纯化后,用SDSPAGE鉴定TIMP3的纯度;用EIA和Westernblot检测TIMP3的含量;用免疫荧光测定法检测TIMP3对MMP1的抑制活性。结果:人胎盘来源的TIMP3由相对分子质量(Mr)为24000的非糖化蛋白和Mr为27000的糖化蛋白组成。非糖基化的TIMP3对MMP1的IC50为1.1×10-10mol/L,糖基化的TIMP3为1.2×10-10mol/L,显著高于重组的TIMP3(IC50为8×10-8)。结论:人胎盘来源的TIMP3由Mr为24000的非糖基化蛋白和Mr为27000的糖基化蛋白两部分组成,二者对MMP1均具有明显的抑制作用。
OBJECTIVE: To isolate and purify tissue inhibitor of metalloproteinase 3 (TIMP3) from human placenta and evaluate its inhibitory effect on matrix metalloproteinase-1 (MMP1). Methods: Insoluble protein was extracted from placental homogenate from which most water-soluble proteins were removed by using Tris HCl buffer (pH 8.0) containing 4 mol / L urea. After purification by CM52 cation exchange resin and Sephacryl S200 gel filtration two-step column chromatography, the purity of TIMP3 was identified by SDSPAGE. The content of TIMP3 was detected by EIA and Western blot. The inhibitory activity of TIMP3 on MMP1 was detected by immunofluorescence assay. Results: Human placenta-derived TIMP3 consisted of non-glycated protein with molecular weight (Mr) of 24000 and glycated protein with Mr of 27000. The non-glycosylated TIMP3 had an IC50 of 1.1 × 10-10 mol / L for MMP1 and 1.2 × 10-10 mol / L for glycosylated TIMP3, significantly higher than that of recombinant TIMP3 (IC50: 8 × 10-8). CONCLUSION: Human placenta-derived TIMP3 consists of 24000 non-glycated proteins and 27000 glycoproteins, both of which have significant inhibitory effects on MMP1.