The Effect of Smad4 and Wnt3a on the Spectrum of miRNA of MSCs

The Objective: purified MSCs stimulated with Wnt3a and smad4 observe the changes of miRNA profiling, looking for osteogenesis miRNA experimental basis. Methods: SD rats were chosen two weeks after birth, sterile under the femur bone marrow cells were isolated and cultured primary bone marrow mesenchymal stem cells; draw its growth curve; detected in the culture of the sixth generation of the flow cytometry has purified bone marrow mesenchymal stem cells. The pcDNA3.1-smad4 and the shuttle plasmid pGStrack-HB assembled rely on homology shuttle plasmid pGStrack-smad4 the transfer to pGSadeno adenovirus expression vector packaging recombinant adenovirus; HEK 293, linearization, complete pGSadeno-smad4 of building ; PCR verification of recombinant adenovirus generation. To the rAd5-smad4 transfection and Wnt3a stimulated culture of MSCs laser confocal observation Wnt3a smad4 group β-cat/Smad4 aggregation phenomena in MSCs. Trizol extraction of total RNA in the cells, making chips with LuxScan 10K / A dual-channel laser scanner scans the chip. Using LuxScan3.0 image analysis software of chip image analysis. Significance Analysis of Microarrays detect impact the Wnt3a smad4 MSCs expression of miRNA Pu, selected differentially expressed genes. And observation of MSCs survival time and apoptosis results: After separation cultured primary bone marrow mesenchymal stem cells after several passages, cells gradually purified, and cells in the early multi-form of colony growth, 6th generation cells showed morphological uniform spindle-shaped, evenly distributed. Flow cytometry, the results show that the volume homogeneous cell populations, CD31, CD34, CD45 positive rate were 5.67%, 4.31%, 4.42%, CD90, CD44, CD71 positive rates were 97.17%, 98.63%, 95.86%. Culture isolates of the test cells have antigenic properties of all MSCs, normal cell growth rate and morphology. Shuttle plasmid, linear cutting and packaging build rAd5-smad4; the the rAd5-smad4 in smad4PCR fragment size of approximately 1.8kb results are in line with the expected, indicating that the recombinant adenovirus the rAd5-smad4 packaging success. The rAd5-smad4 transfection and Wnt3a stimulated cultured MSCs, the microarry detected hsa-miR-122a, 638,494,450 upregulation; confocal laser display MSCs within the β-cat/Smad4 of polymerization. After transfection, and Wnt3a joint stimulus] Ad5-smad4 MSCs can survive for two weeks / generation; apoptosis rate of 0.02%. The MSCs without passaged long-term survival. The rate of apoptosis in normal cells Conclusion: This test isolated and cultured the cells with the antigenic characteristics of all MSCs, normal cell growth rate and morphology. Shuttle plasmid, the linear cutting and packaging cell line to complete the rAd-smad4 the Construction and Expression; Wnt3a transfected the rAd-smad4 achieve the purpose of genetically modified joint MSCs, MSCs can survive for two weeks / generation; apoptosis rate of 0.02% . Joint stimulation of MSCs high expression of hsa-miR-122a, 638,494,450. This provided the experimental basis for looking for osteogenesis miRNA.