OBJECTIVETo explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro.

METHODSHuman cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods.

RESULTSThe number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a number of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry.

CONCLUSIONMSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells.

Antigens, CD ; analysis ; Antigens, Differentiation, B-Lymphocyte ; analysis ; Cell Count ; Cell Differentiation ; Cell Division ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Mesoderm ; chemistry ; cytology ; Neurofilament Proteins ; analysis ; Neurons ; cytology ; Phosphopyruvate Hydratase ; analysis ; Receptors, Transferrin ; Stem Cells ; chemistry ; cytology

OBJECTIVEThe conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. The present study aimed to identify novel citrullinated proteins in 10 tumor cell lines by 2-D Western blotting (2-D WB).

METHODSTwo identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ten cultured human tumor cell lines: ECA(esophageal cancer cells), HEPG2 (hepatocellular carcinoma cells), SKOV3 (ovarian cancer cells), MCF-7 (breast cancer cells), H292 (lung mucoepidermoid carcinoma cells), HeLa (cervical cancer cells), Lovo (colon cancer cells), OS-RC (renal cell carcinoma cells), PANC-1 (pancreatic cancer cells), and SGC (gastric cancer cells). The expression profiles on one 2-DE gels were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in the tumor cell lines.

RESULTS2-D WB and mass spectrometry identified citrullinated ENO1 (α-enolase), HSP60 (heat shock protein 60), KRT8 (keratin 8), TUBB (tubulin beta), TCRβ (T cell receptor β chain), VIME (vimentin) and PDI in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumor cell lines.

CONCLUSIONThe citrullination of proteins ENO1, HSP60, KRT8, and TUBB suggests a new mechanism in the tumorigenic process.

Blotting, Western ; Cell Line, Tumor ; Citrulline ; metabolism ; Female ; Humans ; Immunoprecipitation ; Mass Spectrometry ; Phosphopyruvate Hydratase ; Vimentin

Objective To investigate alterations in the microtubule-associated protein 2 (MAP-2) of neurons in Wistar rats and the effect of nimodipine (Nim), D-2-amino-5-phosphonovaleric acid (D-AP-5) and mild hypothermia on neuronal MAP-2 following fluid percussion injury (FPI). Methods Alterations of MAP-2 in Wistar rat neurons following FPI were measured by a confocal laser scanning microscope using MAP-2 immunofluorescence staining as a MAP-2 indicator. Results MAP-2 immunofluorescence staining was limited to the cell bodies and dendritic compartments of neurons and more intense in dendrites than in cell bodies. The loss of MAP-2 was marked at 3 h post trauma ( P ＜ 0.01 ), and reached a maximum at 48 h post-trauma. Afterwards, fluorescence recovered partly at 72 h post-trauma. The application of Nim markedly reduced the loss of MAP-2 immunoreectivity within 1 h post-trauma ( P ＜ 0.01 ), and the application of D-AP-5 markedly reduced the loss of MAP-2 immunoreactivity within 10 h post-injury ( P ＜ 0.01 ). The application of mild hypothermia decreased the loss of MAP-2 immunoreactivity within 1 h post-injury (P＜ 0.05). Conclusions The partial recovery of fluorescence at 72 h post-trauma indicate that the partial structure of the neuronal microtubules can be repaired by itself. Nim, D-AP-5 and mild hypothermia reduce the degradation of MAP-2 by different mechanisms. The treatment of neuronal cytoskeleton degradation following FPI must employ multiple therapeutic approaches.