Differentiation Of Rabbit BMSCs Into Neuron-like Cells Which Were Tagged With EGFP Gene

Objective To obtain rabbit bone marrow mesenchymal stem cells(BMSCs) which arestable expressed exogenous enhanced green fluorescent protein(EGFP) gene throughtransfecting MmiR3407-MR03recombinant plasmid containing EGFP gene byliposome transfection method, and make them differentiate into neurons with ciliaryganglion neurotrophic factor(CNTF) in order to observe the influence of EGFP gene torabbit BMSCs’s biologicall characteristics and differentiation characteristics intoneurons in vitro, so as to provide more effective tracer methods for transplantation ofBMSCs in vitro.Methods Choosed new born New Zealand white rabbits, propagated purify BMSCsby density gradient centrifugation and adherent method with their bone marrow.Usedimmunocytochemistry method to detect CD34, CD44, CD54antibody’s expression inrabbit BMSCs. Extracted plasmid DNA according to the high purity plasmids littlemention of kit instructions. Determined the best screening G418concentration, usedcationic liposome mediated transfection method to transfer the3-5generations ofrabbit BMSCs and calculated the transfection efficiency. Obtained stable rabbitBMSCs which were expressed EGFP gene by G418flitted and Observed the cellgrowth. Choose P3-5EGFP-BMSCs and P3-5BMSCs, divided them into four groups:EGFP-BMSCs induced group, BMSCs induced group, EGFP-BMSCs blank groupand BMSCs blank group. EGFP-BMSCs induced group and BMSCs induced groupwere used10ng/mlbFGF preliminary induced24h firstly, then use10ng/mlCNTFinduced12h; EGFP-BMSCs blank group and the blank group of BMSCs were nospecial treatment. Observed changes in cells morphology, detected cells activity byMTT method and detected nest protein (nestin) and neuron specific enolase (NES)expression by immunocytochemistry method.Compared cells morphology,cellsviability and differences of neuronal specific marker-positive cells rate between the Induction groups,the blank groups and the cells before and after induction.Results1.The original generation of rabbit BMSCs growed adherent, they looked likespindle and whirlpool. After passage,the cells tended to be consistent gradually,cellfull,and more orderly arrangement. Passed to10generation, cell aged.2.Immune cell chemical technology results showed that CD44, CD54werepositive expression, CD34expression is negative, according with BMSCs immunephenotypic characteristics.3.12h after transfection, a small amount of cells expessed green fluorescentwhich were observed under the fluorescence microscope;48h aftertransfection,transfection rate was the highest, nearly30%. Little change was happenwithin the next72h. Obtained stable rabbit BMSCs which were expressed EGFP geneby G418flitted.4. induced12h with CNTF, the cells had two or more slenders, strong refraction,similared neurons in the form, bright green fluorescence is still visible influorescence microscope. MTT showed that:24h,48h and72h, the difference of ODbetween the BMSCs induced group,the EGFP-BMSCs induced group,theEGFP-BMSCs blank group and the BMSCs blank group had not statisticallysignificant (P>0.05).5.Immunocytochemistry test showed that the rate of nestin positive cells and therate of NSE positive cells in EGFP-BMSCs induced group were (49.983.07)%,(48.741.32)%separately; the rate of nestin positive cells and the rate of NSEpositive cells in BMSCs induced group were (48.443.97)%,(47.861.68)%separately; the rate of nestin positive cells and the rate of NSE positive cells in EGFP-BMSCs blank group and BMSCs blank group are all small.The difference ofantibody positive rates between EGFP-BMSCs induced group and BMSCs inducedgroup had no significant statistically (P＞0.05); The difference of antibody positiverates between EGFP-BMSCs blank group and BMSCs blank group had no significant statistically (P＞0.05); The difference of antibody positive rates between BMSCsinduced group and BMSCs blank group had obvious significance statistically (P＜0.05); The difference of antibody positive rates between EGFP-BMSCs induced groupand EGFP-BMSCs blank group had obvious significance statistically (P＜0.05).Conclusion1.By liposome, we can obtain rabbit BMSCs which are expressing EGFP stably，EGFP gene markers may be an effective tracer method for the BMSCs transplantation;2. The rabbit BMSCs labeled with EGFP gene can be differentiated into neuronssuccessfully, which have no effect on their biological characteristics;3. Rabbit BMSCs which are expressing EGFP stably can differentiate intoneuron-like cells in the intervention of ciliary ganglion neurotrophic factor.