The Construction And Biological Detection Of A Targeted Glioma MRI Molecular Probe Based On MMP-2

Objective: Glioma is the most common primary central nervous system tumors, with a high degree of malignancy, easy to relapse, clinical features and poor prognosis. Over the years, glioma’s diagnostic tests has been an important issue, accurate and timely diagnosis is important to control the disease in the late and improve the overall treatment effect. Studies have shown that the high mortality rate of glioma owe to the height of the invasive ability of tumor cells, which is the typical biological characteristics of gliomas [1].Through attaching to the extracellular matrix(ECM), glioma cells then degradated its components gradually, eventually penetrate into the adjacent brain tissue. In this process, as one of the matrix metalloproteinases(MMPs) family, matrix metalloproteinase-2(MMP-2) plays a vital role, it can take part in tumor invasion process by degradate the extracellular matrix(ECM) components and promote angiogenesis mechanisms[2]. Some research have pointed out that the degree of malignancy grade gliomas and the expression of MMP-2 have a close relation, the higher the degree of malignancy, immunohistochemistry showed positive expression of MMP-2 also will be increased [3].Because of its superior soft tissue resolution and contrast ratio, Magnetic resonance imaging has been the preferred brain imaging techniques of glioma’s diagnosis and treatment. Ultrasmall superparamagnetic iron oxide particles(USPIO) as a MRI contrast agent has higher sensitivity and saturation than conventional MRI contrast agents, magnetic relaxation rate is 7-10 times to conventional MRI the contrast agent under the same conditions [4], and its diameter less than 20 nm, which could avoid being phagocytosis by reticular endothelial system(RES). By surface modification, it can be used in cell or tissue imaging with specific molecular [5]and reduce MRI T2 signal intensity [6,7]. Meanwhile USPIO has lower toxicity, it can be degraded after entering the body with normal plasma iron pool then metabolized by cell [8-11].This experimental design to couple the polyclonal antibody of matrix metalloproteinase-2(Ab-MMP-2) and Ultra small superparamagnetic iron oxide particles(USPIO), form a molecular imaging probe Ab-MMP-2-USPIO which has the specificity target function.Then take In vitro tests to detect immunocompetence、cytotoxicity and specific binding ability to the glioma cells of the probe. To explore the feasibility and effectiveness of the probe’s application in malignant glioma’s diagnosis with MRI molecular imaging, provide new method and direction for the diagnosis of glioma.Methods:Part 1. Detect the expression of matrix metalloproteinases-2 in three cell lines.Using immunofluorescence and western blot to detect matrix metalloproteinase-2(MMP-2)’s expression in rat astrocytes(RA)、rat glioma cell line(C6) and one human glioma cell line(U87), respectively;Part 2. Coupling the polyclonal antibody of matrix metalloproteinases-2(MMP-2)’s and ultra small superparamagnetic iron oxide(USPIO) particles by carbodiimide method(EDC).Part 3. Detect the biological activity and specific binding ability with target cell of the synthesis molecular probe.1 Using Western blotting method to detect the immune activity of molecular probe Ab-MMP-2-USPIO;2 Using MTT colorimetry to detect the cytotoxicity of molecular probe Ab-MMP-2-USPIO to normal rat astrocytes(RA) in different concentrations(0, 25, 50, 75, 150, 300, 600 ug/ml).3 Using Annexin V-FITC/PI double staining method to detect the apoptotic role of molecular probe Ab-MMP-2-USPIO to normal rat astrocytes(RA) and rat glioma cells(C6) which concentration is 75 ug/ml.4 Using Prussian blue staining method to detect the Specific binding ability of molecular probe Ab-MMP-2-USPIO to the matrix metalloproteinase-2(MMP-2) in rat glioma cells(C6) which concentration is 75 ug/ml.Results:1 Immunofluorescence showed that there is no specific fluorescence in rat astrocytes(RA) related to MMP-2 protein’s expression, which is highly expressed in rat glioma cell(C6) and human astrocytoma cell(U87) in the cytoplasm; Western blotting results can be seen stripe in rat glioma cell(C6) and human astrocytoma cell(U87) groups, confirmed that MMP-2 protein expressed in this two kind of cell, but in normal rat astrocytes(RA) group was no MMP-2 protein expression detected.2 The test results of the composite molecular probe Ab-MMP-2-USPIO are: Western blotting showed that Ab-MMP-2-USPIO and MMP-2 protein has a high combination degree, and similar to the polyclonal antibody of MMP-2; MTT colorimetry assay showed low cytotoxicity Ab-MMP-2-USPIO has, more than 150ug/ml with 72 h cultured could be figure out significant cytotoxicity after all, the dosage used in subsequent experiments(75ug/ml) for normal rat astrocytes is in a safe level; Annexin V-FITC/PI double staining method showed that 75ug/ml of Ab-MMP-2-USPIO cultured with the normal rat astrocytes(RA) and rat glioma cells(C6) in 48 h and 72 h will not cause significant apoptosis; Prussian blue staining showed compared with normal rat astrocytes(RA), rat glioma cells(C6) cells are able to ingest a lot of Ab-MMP-2-USPIO.Conclusion :The results of in vitro experiments showed that, MMP-2 protein is not expressed in normal rat astrocytes(RA), which meanwhile highly expressed in rat glioma cells(C6) and human glioma cells(U87). The cytotoxicity of synthetic molecular probe Ab-MMP-2-USPIO is small, and apoptosis effect is not obvious. In the meantime, it has a high specific binding capacity with rat glioma cells(C6). Which laid the foundation for in vivo experiments in the future, and can be expected to become a novel targeted nano-MRI contrast agent which could improve the accuracy of MRI diagnostic accuracy in glioma.