论文部分内容阅读
背景:基质金属蛋白酶是一类可促进肿瘤生长和转移的蛋白分解酶家族,其活性可被组织金属蛋白酶抑制因子所抑制。其中组织金属蛋白酶抑制因子3的抑制作用更为重要。目的:旨在从人胎盘中完全分离纯化天然组织金属蛋白酶抑制因子3,并建立组织金属蛋白酶抑制因子3酶联免疫测定方法。设计:单一样本观察。单位:沈阳医学院中心实验室。材料:人胎盘来自沈阳医学院附属奉天医院妇产科(家属知情同意);基质金属蛋白酶1来自富士药品研究所。方法:实验于2001-03/2002-05在沈阳医学院中心实验室完成。①利用4mol/L尿素Tris缓冲液(pH8.0),制备胎盘匀浆液。②匀浆液经CM52阳离子交换树脂层析和SephacrylS-200凝胶过滤二步柱层析。③SDS-聚丙烯酰胺凝胶电泳检测相对分子量及纯度。④Westernblotting鉴定纯化蛋白的性质。⑤免疫荧光法测定组织金属蛋白酶抑制因子3对基质金属蛋白酶1的抑制率。主要观察指标:①聚丙烯酰胺电泳法所示蛋白带相对分子质量。②Westernblot的显色结果。③组织金属蛋白酶抑制因子3对基质金属蛋白酶1的抑制率。④纯化后组织金属蛋白酶抑制因子3的回收率。结果:①人胎盘分离纯化的组织金属蛋白酶抑制因子3分为非糖化和糖化两种,相对分子质量分别为24000和27000两种。②非糖化和糖化的组织金属蛋白酶抑制因子3对基质金属蛋白酶1的抑制活性分别为1.1×1010mol/L和1.2×1010mol/L。胎盘来源的组织金属蛋白酶抑制因子3对基质金属蛋白酶1的抑制活性明显高于重组的组织金属蛋白酶抑制因子3。③二步层析后组织金属蛋白酶抑制因子3的回收率为23.4%。结论:人胎盘胞外基质来源的组织金属蛋白酶抑制因子3是由非糖基化蛋白和糖基化蛋白组成;两种纯化的组织金属蛋白酶抑制因子3对基质金属蛋白酶1具有明显的抑制活性,且显著高于重组金属蛋白酶抑制因子3。
BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that promote tumor growth and metastasis, and their activity can be inhibited by tissue inhibitors of metalloproteinases. Among them, the inhibition of tissue inhibitor of metalloproteinase 3 is more important. OBJECTIVE: To completely separate and purify native tissue inhibitor of metalloproteinase 3 from human placenta and to establish a method for the determination of tissue inhibitor of metalloproteinase 3. Design: Single sample observation. Unit: Shenyang Medical College Center Laboratory. MATERIALS: Human placenta was from Obstetrics and Gynecology Hospital affiliated to Shenyang Medical College (family members informed consent); MMP-1 was from Fuji Pharmaceutical Research Institute. Methods: The experiment was performed at the Central Laboratory of Shenyang Medical College from March 2001 to May 2002. ① using 4mol / L urea Tris buffer (pH8.0), preparation of placental homogenate. ② homogenate by CM52 cation exchange resin chromatography and Sephacryl S-200 gel filtration two-step column chromatography. ③ SDS-polyacrylamide gel electrophoresis detection of relative molecular weight and purity. ④ Westernblotting identification of purified protein properties. ⑤ Immunofluorescence assay of tissue inhibitor of metalloproteinase 3 on matrix metalloproteinase 1 inhibition rate. MAIN OUTCOME MEASURES: (1) Polyacrylamide electrophoresis shows the molecular weight of the protein band. ② Western blot results of color. ③ tissue inhibitor of metalloproteinase 3 matrix metalloproteinase 1 inhibition rate. ④ purification of tissue inhibitor of metalloproteinase 3 recovery. Results: ① The tissue inhibitor of metalloproteinase 3, which was isolated and purified from human placenta, was divided into two types, non-glycated and glycosylated, with relative molecular mass of 24000 and 27000 respectively. ② The inhibitory activity of non-glycosylated and glycated tissue inhibitor of metalloproteinase-3 on matrix metalloproteinase-1 was 1.1 × 1010mol / L and 1.2 × 1010mol / L, respectively. Placental-derived tissue inhibitor of metalloproteinase 3 has a significantly higher inhibitory activity on matrix metalloproteinase-1 than recombinant tissue inhibitor of metalloproteinase-3. The recovery of tissue inhibitor of metalloproteinase 3 after two-step chromatography was 23.4%. CONCLUSION: Tissue metalloproteinase 3, an extracellular matrix of human placenta, is composed of non-glycosylated protein and glycosylated protein. The two purified tissue inhibitors of metalloproteinase 3 have obvious inhibitory activity on matrix metalloproteinase 1, And significantly higher than that of recombinant metalloproteinase inhibitor 3.