| Objective1To set up the method of separating mesenchymal stem cells(MSCs)from the full-term pregnantwoman umbilical cord,and the way of proliferation in vitro.To study the biologic characteristicsand phenotypic characteristics of MSCs in vitro,preparing for the application of MSCs in thefollowing study.2To explore the feasibility of labeling MSCs with Gd-DTPA,and to analyze the effect of thebiologic characteristics of the labeling stem cells.3To transfect Gd-DTPA into stem cells with different transfection reagents,and to find thehightest transfecting efficiency of transfection reagents,laying the experimental foundation forMR imaging rule of magnetic labeling stem cells.Methods1In sterile condition,obtain the umbilical cord sample from healthy full-term fetus,apply tissueadherent method to separate the umbilical cord mesenchymal stem cells. MSCs are cultivatedwith10%fetal bovine serum in DMEM/F12medium.2Detect cell surface markers with flow cytometry;Observe the morphology of cells by opticalmicroscope;Counting method assays cell value-added and depicts the growth curve; Induce theMSCs to differentiate into adipocytes and osteoblasts and to verify the differentiation capacity.3To transfect Gd-DTPA into mesenchymal stem cells with transfection reagents,then are imagedby a clinical1.5T MRI imaging system and observe the signal intensity.4Counting method assays magnetic labeling cell value-added；Induce the magnetic labeling cellto differentiate into adipocytes and osteoblasts,Observe the effect of biologic characteristics ofmagnetic labeling stem cells.5Transfect Gd-DTPA into mesenchymal stem cells with Calcium phosphate, Effectene andLipofectamine2000; Test the signal intensity by MRI, measure the Gadolinium concentration within the cells by inductively coupled plasma atomic emission spectrometry；find the hightesttransfecting efficiency of transfection reagents by statistical analysis.Explore the lowest cellsquantity and Gd with cells passaged variation that can be observed.Results1Primary cells are got by tissue culture after two weeks,the cell are fusiform, whirlpool-likegrowth. Growth rate was significantly increased after passage, the second passage of MSCs aremorphologically homogeneous. Cell growth curve shows that cell growth from4days to enterthe logarithmic growth phase.Most of the third passage of MSCs express CD29、CD44、CD90、CD105, but hematopoietic cell surface markers CD45and allograft rejection are closely relatedsurface markers CD40, HLA-DR were negative,which are identified as MSCs. mesenchymalstem cells can be induced to adipocytes and osteoblasts in vitro.2Three transfection agents can successfully transfected with Gd-DTPA into stem cells, cellsshow high signal in T1WI by MR scanning in vitro.3Transfected with Gd-DTPA stem cells′proliferation and differentiation capacity were notaffected.4Test the signal intensity by MRI and measure the Gadolinium concentration within the cells byinductively coupled plasma atomic emission spectrometry，find that effectene is the besttransfecting efficiency of transfection reagents.The lowest magnetically labeled cells quantitywere5×10~4, Gd with cells passaged variation that can be observed. As the cells passaged, thenumber of Gd particles inside the cells and the cell′s signal intensity in T1WI were graduallydecreased.Conclusions1hUCMSCs were successfully isolated and cultured from the umbilical cord by tissue adherentmethod, can be stable subculture and obtain more pure MSCs.2hUCMSCs have biological characteristics of mesenchymal stem cells and differentiationcapacity by inducing to adipocytes and osteoblasts in vitro.3Application of Gd-DTPA-labeled human umbilical cord mesenchymal stem cells is feasible，Labeled stem cell proliferation and differentiation potential were not affected.41.5T clinical MRI can trace stem cells transfected with Gd-DTPA in vitro. The cell signalintensity is gradually decreased with the cells passaged.Magnetically-labeled stem cells by MR tracing lasted about12days in vitro. |
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