| ObjectiveThe study was to observe the expression of human NT-3 gene and EGFP gene and whether induce BMSCs'differentiation to neural cellula by cationic liposome medicated eukaryotic co-expression vector(pIRES2-EGFP-NT-3) With the reporter gene-GFP was constructed transfect rat bone marrow stromal cells.Materals And Methods1.Isolation and culture of BMSCsExperiments were carried out on male Wistar rats (body weight,100-150 g). After the thiqhbones was dissected out in a sterile condition under anesthesia,the bone marrow was used for cell culture and separation by whole bone marrow adherence mothod.The BMSCs was purified through subculturing and counted for use.2.Identification of BMSCsTo characterize these cells in an integrated approach, flow cytometry was proformed to identify specific antigens'expression, such as CD34, CD90. We also make the BMSCs induced osteoblastic differenciation and adipocytic differenciation by incubation with different culture media.3.Cationic liposome medicated NT-3 gene transfect BMSCsTransfection of the P3 BMSCs was processed by cationic liposome in 6-well culture plate. BMSCs was divided into non-transfected group(A), empty vector transfection group(B), transfection group(C) randomly, then the transient expression was detected 48 hour after transfection.4. Target detection(1) Observation and record of GFP's expression in each group under inverted fluorescence microscope.(2) Expression of NT-3 and NSE mRNA in each group was detected by RT-PCR 48h after transfection.(3) Expression of NT-3 protein in each group was detected by Western-blot 48h after transfection.Results1. The morphological observation and identification of rat BMSCs3 days after Primary cells inoculation, A large number of dead cells suspended can be seen under inverted microscope, with a round shape, mostly known as hemocyte. After change the culture media, adherent cells with colony-like growth distributed unevenly, with a shape of Fusiform, triangular numerously. About 8-10 days to reach 90% of cell fusion. As subculture goes, BMSCs'degree of purity get higher an higher. A great number of long-spindle cells of uniform adherent growth, clustered arrangement, and get into the logarithmic growth phase after 24h passage. P3 cells were gyrate growth homogeneously, with a fundamental consistent long-spindle shape.Rat BMSCs were identified with CD34 and CD90 with FCM. Most cells were negative in CD34 and positive in CD90, the positive rate of CD34 is 0.43%; the positive rate of CD90 is 96.77%. ALP was strongly positive for BMSCs by Gomori staining after osteogenic induction. Different red lipid droplets were observed in cytoplasm of BMSCs by oil red O staining after adipogenic induction.2. Expression of GFPCells of each group were observed under inverted fluorescence microscope. The GFP can be seen both in B and C group, compared to A group (non-transfected group).3. RT-PCR resultsThe expression NT-3 mRNA in C group was significantly higher than A and B group, almost no expression of NT-3 mRNA in A and B group. The expression of NSE mRNA was also significantly higher than A group.4. Western-blot resultsThe expression of NT-3 protein was significantly higher than A and B group, while a weaker expression both in A,B group.Conclusion 1.BMSCs were successfully isolated and cultured by whole bone marrow adherence method. According to the adherence character, it can be bring to achieve the requirements of experiment after purified by passage.2.Eukaryotic co-expression vector (pIRES2-EGFP-NT-3) can be transfected into rat BMSCs successfully by lipofectamin2000.3.The growth of transfected BMSCs was in good condition, and the expression of target gene NT-3 and trace gene GFP was also fine.4.NT-3 can promote the differentiation to neuron-like cells of BMSCs after transfected into BMSCs.
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