| Objective: 1)To study the potential of differentiation of human adipose derived stem cells(hASCs) into smooth muscle cell; 2) To investigate the role of miR-145 in regulating the differentiation of the hASCs into smooth muscle cell; 3) Determine the role of Kruppel like factor 4(KLF-4) in the smooth muscle differentiation of hASCs; 4) To clarify the role of Ca2+/calmodulin-dependent protein kinase II ?(CaMKII?) in modulation of hASCs differentiate into smooth muscle cells. Methods: 1) Human adipose derived stem cells were isolated from lipoaspirate, cultured and expanded to passage 3. Cells at passage 3 were induced by combined stimulation of transforming growth factor beta 1(TGF-?1) and bone morphogenetic protein 4(BMP4). Expression of smooth muscle cell specific markers including a-SMA,SM22 a,Calponin and SM-MHC were detected by quantitative real-time PCR, western blot and immunofluorescence staining,respectively; 2) Expression of miR-145 in induced hASCs was detected by quantitative real-time PCR after 7 days of combined stimulation by TGF-?1 and BMP4. Mimics and inhibitors of miR-145 were synthesized, and transfected into hASCs respectively. After transfection,expression of a-SMA,SM22 a,Calponin and SM-MHC were determined; 3) Furthermore, association between miR-145 and target genes was investigated by the luciferase reporter gene technology.miR-145 target genes were predicted by searching biological information system. Association between miR-145 and target gene was determined by the luciferase reporter gene technology, target gene expression were examined under overexpression and knock-down of miR-145 respectively. hASCs were tranfected with si-KLF-4 and expression of a-SMA,SM22 a,Calponin and SM-MHC were observed; 4) The expression of Ca MKII gamma was detected in the smooth muscle differentiation of hASCs. The overexpression and silent vector of CaMKII? were synthesized and transfected into hASCs, transfection efficiency was detected. The hASCs were induced to smooth muscle cell after transfection, a-SMA,SM22 a,Calponin and SM-MHC expressions were detected by quantitative real-time PCR, western blot and immunofluorescence staining, respectively. Results: 1) The hASCs at passage 3 showed elongated fibroblast like morphology under inverted microscope. CD13, CD34, CD44, CD49 d, CD90, CD105 and CD166 were positive expressed, and hematopoietic cells and endothelial cells markers including CD14, CD45 and CD31 were negative expressed. hASCs acquired a spindle morphology and grew in a ??hill and valley?? pattern after smooth muscle cell differentiation and expressed smooth muscle cell specific markers: a-SMA, SM22 a, calponin and SM-MHC; 2) The expression of miR-145 was increased when hASCs treated with TGF-?1 and BMP4. After transfected with miR-145, the expression of smooth muscle cell specific markers were significantly increased, while suppression of miR-145 caused the opposite results; 3) KLF-4 was identified as a target gene of miR-145. The expressions of KLF-4 was decreased in the smooth muscle differentiation of hASCs. KLF-4 was downregulated by miR-145, up-regulated by miR-145 inhibitor. Suppression of KLF-4 upregulated the expression of smooth muscle cell specific markers during smooth muscle differentiation; 4) The expression of CaMKII? were significantly increased during the smooth muscle cell differentiation of hASCs. Over expression of Ca MKII ? increased the expression of smooth muscle cell specific markers, and silence the CaMKII? led to inhibition of the smooth muscle differentiation. Conclusion: 1) It is easier to acquire hASCs from adipose tissue and growth well in the basic culture condition. hASCs is an ideal cell sources of tissue engineering; 2) hASCs can be induced to smooth muscle cells by combined stimulation with TGF-?1 and BMP4; 3) miR-145 plays a positive regulatory role in smooth muscle cells differentiation of hASCs, and KLF-4 was a target gene of miR-145; 4) CaMKII? promotes the differentiation of the hASCs into smooth muscle cell. |
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