The Effect Of Smad4 And Wnt3a On The Spectrum Of MiRNA Of MSCs

Objictive:Combined stimulation of purified MSCs with smad4 and Wnt3a, observed changes in miRNA spectrum, to provide experimental basis for searching for related miRNA of osteogenesis.Methods:Two weeks old SD rats were choosed, gain the bone marrow cells of femoral. Then isolated and coltured the bone marrow mesenchyal stem cells. Drawed the curve of growth. Detected the purified MSCs with sixthed generation by Flow cytometry. Assembled pcDNA3.1-smad4 to pGStrack-HB, then transferred pGStrack-smad4 to pGSadeno which was adenovirus carrier. Recombined adenovirus by transfecting HEK293 cell, and here completed the construction of adenovirus of pGSadeno-smad4. Then validated the recombinant adenovirus by polymerase chain reaction. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to cultured the MSCs, confocal observation of phenomenon aggregate ofβ-cat/Smad4 in MSCs, which were stimulated Wnt3a and smad4. Extracted the total RNA by using Trizol method, producted and scanned chips with LuxScan 10K/A of dual channel. Images were analyzed by Image analysis software LuxScan3.0. Used Significance Analysis of Microarrays to detect miRNA expression profile of MSCs, which were stimulated Wnt3a and smad4, selected the different expression of miRNAs. And observed the survival time and apoptosis of MSCs.Results:The bone marrow mesenchymal stem cells gradually purified after isolated and cultured primary MSCs were passaged for many generations, in the early stage of growth the cells formed colonies, the cells were uniform shape and distribution after the sixth generation. And flow cytometry showed that cell populations had homogeneous volume, the positive rates of CD31, CD34, CD45 were 5.67%,4.31%, 4.42%, and of CD90, CD44, CD71 were 97.17%,98.63%,95.86%. These results show that the isolated and cultured cells had the same antigenic characteristics of MSCs, growth rate and morphology were normal.Constructed rAD5-smad4 through the shuttle plasmid and packaging. PCR fragments size of smad4 rAd5-smad4 were about 1.8kb, the results are in line with expectation, which indicated that adenovirus rAd5-smad4 packaged successfully. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to cultured the MSCs, detected hsa-miR-122a,638,494 and 450 upregulation by microarry. Confocal observed the phenomenon aggregate ofβ-cat/Smad4 in MSCs. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to the MSCs, MSCs could survive for 2 weeks of generation with 0.02% of apoptosis rate. MSCs had normal rate of apoptosis.Conclusion:isolated and cultured primary MSCs had the same antigenic characteristics of MSCs, growth rate and morphology were normal. rAD5-smad4 was constructed successfully. Combined the stimulation with Wnt3a and transfection of rAd-smad4 to the MSCs, and achieved the purpose of genetically modified to MSCs. We found hsa-miR-122a,638,494 and 450 were upregulation, which provided experimental basis for searching for related miRNA of osteogenesis.