人胎盘来源的金属蛋白酶抑制因子-3的分离纯化及性质研究

Purification and Characteristic Study of Native Tissue Inhibitors of Metalloproteinase-3 from Human Placenta

目的 首次从人胎盘中分离纯化了天然型TIMP 3 ,并建立了TIMP 3酶联免疫测定方法。方法 人胎盘来源的TIMP 3的纯化 ,首先利用 4Mol/L尿素Tris缓冲液 (pH8.0 ) ,在除去多数水溶性蛋白的胎盘匀浆液中提取难溶性蛋白 ,然后经过CM5 2阳离子交换树脂和SephacrylS 2 0 0凝胶过滤二步柱层析。结果 SDS聚丙酰胺凝胶电泳显示最后分离结果 ,分别为 2 7KD和 2 4KD的两条蛋白带。Westernblotting结果表明 ,分子量为 2 7KD蛋白为N末端糖化的TIMP 3、分子量为 2 4KD的蛋白为非糖化的TIMP 3、两者的比例约为 1∶3。对比尿素抽提液TIMP 3的纯化产率为 2 4 %,纯化倍数约为 3 2 0 0倍。结论 纯化的天然型TIMP 3对MMP 1有明显的抑制活性。非糖化的TIMP 3IC50 为 1 .1× 1 0 - 1 0 M ,糖化的TIMP 3IC50 为 1 .2× 1 0 - 1 0 M ,显然比重组TIMP 3对MMP 1的抑制活性要高许多 (rTIMP 3的IC50 为 1 .2× 1 0 - 8M)。研究表明 ,TIMP 3对某些肿瘤转移有明显的抑制效应。此项研究对进一步阐明MMPs和TIMPs在机体内平衡关系的改变对肿瘤浸润转移的影响作用有着重要的意义。

Objective The native tissue inhibitor of metalloproteinase-3(nTIMP-3) was purified from human placenta and nhTIMP-3 Enzyme Immunoassay was founded. Method nhTIMP-3 was purified by using an urea extraction technique followed by successive chromatography in CM52 cellulose and Sephacryl S-200 column. Results The pooled fractions gave 98% purity of nhTIMP-3 with molecular weight 24Kda in SDS-PAGE and gave 3200purified fold and 23.4% recovery. And also gave another protein band with mW27Kda in SDS-PAGE. Western blotting verified that protein with 24Kda is unglycosylated nhTIMP-3, 27Kda is glycosylated nhTIMP-3. Conclusions It means that nhTIMP-3 is one kind of protein with partial glycosylation. Both of nh TIMP-3 are able to be detected by EIA(Enzyme Immunoassay). The purified nhTIMP-3 evidently inhibited Matrix Metalloproteinases-1 (MMP-1) activity and its inhibition was quantitatively more than purified rhTIMP-3.