MR Monitor Acute Cell Rejection By Imaging CD4+T Lymphocyte Which Labled With SPIO Bioprobe

Objective: to study whether superparamagnetic nanoparticles, used clinically for specific magnetic sorting, can be used as a specific magnetic cell label for in vivo cell visualization by image specific labeling CD4 T lymphocyte in vitro and vivo in mice. Materials and Methods:1. In vitro:Human CD4~+ T lymphocyte were selected from human peripheral blood via automated magnetic cell sorter and labeled by CD4~+ monoclonal antibody (mAb)-coated microbeads. After labeling , the CD4 lymphocyte were examined with flow cytometry, Prussian blue staining, immuocytochemistry, electron microscope , and characters were detected including trypan blue exclusion test,MTT,and apoptosis detection . Atom pectro photometer was used to determine the effective labeing concentration of CD4 micrbeads; ex vivo of MRI for exploring the efficiency, of labeling.2. In vivo:the mice models which received the pig islets under left kidney sub-capsule were classified to two groups ,that is experimental group who injected labled CD4~+T lymphocyte by i.p while control group who taken injection of non immunomagnetic beads. After injection of 3, 7, 14days, 1.5T MRI was performed with spin echo T1-weighted imaging and T2-weighted imaging to scan the islets grafts .Animals were sacrificed as long as the singal losses on the religion of grafts were observed by MRI .Pathobiology, Prussian blue , immunohistochemistry were performed for determining the infiltrationof labeled CD4T lymphocyte in islets grafts.Results1. In vitro:1.1 Flow cytometry, Prussian blue staining, immuocytochemistry, and electron microscope have confirmed that SPIO are conjugated at the surface of CD4~+ T lymphocyte;1.2 Trypan blue exclusion test, MTT, and apoptosis detection showed that this mothod of labling isn't harmful to the cells' vitality and proliferation.1.3 The minimal amount of cells which could be detectable forT2-weighted imaging was 5×10~6/ml, while 10~7 /ml demostrated the significant signal loss with same sequce;there was no remarkable signal changes along with the decrease of labeled cells; Meantime,MR with Tl-weighted imaging couldn't detect the signal changes of laled cells;10~7/ml labled cells provided sifficient signal loss even after cultured three days and tnree times elution by PBS.2. In vivo: The infiltration of CD4~+ T lymphocyte with nano bioprobe at the rejection site can be observed noninvasively with MRI in 8 of experimental mice. In the contrary, there was no any signal changes on the area of islets grafts in the control animals. The data obtained from MR correlated with histological findings.Conclusion: In our study we confirmed that CD4 microbeads pan be used as a magnetic label (a highly specific MR contrast agent); they provide sufficient MR contrast although the average iron content per cell is an order of magnitude lower than in the case of cell labeling with Endorem and other contrast agents that enter the cell. The fact that microbeads coated with different commercially available antibodies can bind to specific cell types opens extensive possibilities.