Constructing A Plasmid Pegfp-C₁/Gdnf To Studing The Function Of Differentiation For Bone Marrow Mesenchymal Stem Cells

Part Ⅰ CONSTRUCTING A PLASMID PEGFP-C1/GDNFObjectives:To Clone rat glial cell line-derived neurotropic factor (GDNF) gene and to reconstruct the pEGFP-C1/GDNF.Methods:Nerual tissue of one-week old SD rat was used to extract total RNA. A636bp positive GDNFcDNA band was obtained by RT-PCR.This fragment was clone into the pEGFP-C1.The cloned fragment was sequenced and identified by restricting enzymes.Results:RT-PCR product was a636bp specific segment. Analysed by restricting enzymes digestion and DNA sequencing, the pEGFP-C1/GDNF recombination was4.7kb and636bp. The DNA sequencing revealed that GDNF cloning was successful.Conclusion:The pEGFP-Ci/GDNF recombination could be used in further research. Part Ⅱ STUDING THE FUNCTION OF DIFFERENTIATION FOR BONE MARROW MESENCHYMAL STEM CELLSObjeetive:To prove that there were glial cells derived neural factor (GDNF) Protein expression in the bone marrow mesenchymal stem cells (BMSCs) after The pEGFP-Ci/GDNF were transfected to BMSCs by gene transfer technology,and the expressed GDNF protein can induce the BMSCs into nerve cells.Methods:1. Choose training forth generation BMSCs which have a high purity and a good form uniformity tested by the flow cytometric detection, using Lipofectamine2000as the carrier transfect pEGFP-Ci/GDNF to BMSCs.2. Make a control study between pEGFP-Ci/GDNF transfection groups, the empty carrier groups and without transfection groups.3.In12hours,24hours,48hours,72hours and168hours, to observe fluorescent expression, cells form and the growth of change by a fluorescent microscope.4. The exogenous gene mRNA expressions of three groups were tested by RT-PCR.5.Expression of nestin were identified by immunocytochemical technique.Results:The pEGFP-C1/GDNF transfection group visible green fluorescent expression in24hours to a week, the strongest fluorescence appeared in48hours, and then gradually abate.after week, only visible weak fluorescence; Empty carrier group visible fluorescence expression, but without transfection group have no fluorescence expression. IN pEGFP-C1/GDNF transfection group,12hours, the BMSCs appear distortion At high magnification, individual cells appear unipolar, bipolar neuron-like cells.48hours,deformation of celsgradually increased, the structure is clear, MSCs presented dipolar, multipolar and pyramidal typical neuron morphology, the Cells bumps can be visibled, adjacent cells forming synaptic connection.168hours, the cells body shrinked, some cells appear karyopyknosis, the nuclear cracking, apoptosis. Empty carrier group and without transfection group have no positive expression.The results show that transfection group have GDNFmRNA expression in BMSCs which tested by RT-PCR; Empty carrier group and without transfection group have no positive expression.In24hours and48hours,there were nestin positive expression in pEGFP-C1/GDNF transfection group,the control groups have not nestin positive expression.Conclusion:Restructuring plasmid pEGFP-C1/GDNF were successfully transfected to BMSCs and obtained GDNF protein expression, the expression GDNF protein has the potentiality to induce the MSCs differentiate into neuron-like cells, and can promote it growth.