Effect Of Low Intensity Pulsed Ultrasound On Proliferation And Osteogenic Differentiation In SD Rat Adipose-derived Stem Cells And The Possible Osteogenic Mechanisms

Part ⅠDisassociation, culturing and identification of SD rat ADSCsObjective To obtain mesenchyme stem cells from adipose tissue of Sprague-Dawley rat, and to supply seed cells for follow-up study of tissue engineering.Methods1. Some adipose tissue was mechanically separated from inguinal region of SD rat. Some cells were collected through enzyme digestion and centrifugation.2. The collected cells were cultured after sorted by immunomagnetic beads sorting system; morphological characteristics of these cultured cells were surveyed and cell surface markers were detected.Result1. The fibroblast-like cells were checked out in culture dish; these cells could stably proliferate and maintained same morphological characteristics after passaged.2. The surface markers of these cells were considered in consistence with mesenchyme stem cells as demonstrated by flow cytometer.Conclusion Using mechanical separation binding to enzyme digestion and immunomagnetic beads sorting system, adipose-derived stem cells could successfully be obtained. These stem cells could be cultured in vitro, supply seed cells for follow-up study of tissue engineering. Part Ⅱ Effect of LIPUS on proliferation and osteogenic differentiation of SD rat ADSCsObjective To explore the effect of LIPUS on proliferation and osteogenic differentiation of SD rat ADSCs.Methods1. The cell cycle distribution was respectively detected in the cells cultured1d,2d,3d,4d, or5d of the negative group and LIPUS stimulated group using flow cytometer, the proliferation indexes were counted.2. The cells cultured Id,3d,5d, or7d in negative group and LIPUS stimulated group were respectively counted using CCK-8reagents-box.3. Alkaline phosphatase (ALP) activity of cells cultured7d or14d were determined in four different groups(including negative control, LIPUS stimulated group, osteoinduction group and LIPUS stimulated in combination with osteoinduction group); after cultured21d, Calcium nodule formation were detected by Von kossa staining in four different groups.4. Expression of osteogenic-related Genes (including Runx2, ALP, OCN, BSP and Coll Ⅰ) of cells cultured1d,3d,5d, or7d were checked by RT-PCR in negative group and LIPUS stimulated group.5. Expression of osteogenic-related proteins (Runx2, BSP) of cells cultured7d or14d were measured by Western blot in negative group and LIPUS stimulated group.Results 1. The cell proliferation indexes at4d or5d in LIPUS stimulated group were higher than that of the negative group.2. The degree of cell proliferation at3d,5d or7d in LIPUS stimulated group was accelerated compared to that in negative group.3. Alkaline phosphatase(ALP) activity of cells cultured7d or14d in LIPUS stimulated group were higher than that in negative group, but lower than osteoinduction group or LIPUS binding to osteoinduction group); after cultured21d, Calcium nodule formation emerged in LIPUS stimulated group, but the quantity of Calcium nodule were relative few compared to that in osteoinduction group or LIPUS binding to osteoinduction group.4. Expression of Runx2Gene of cells cultured3d,5d or7d in LIPUS stimulated group were up-regulated, and5d or7d later OCN, BSP were also up-regulated, but Coll Ⅰ remained unchanged.5. Expression of Runx2or BSP proteins of cells cultured7d or14d were up-regulated in LIPUS stimulated group, up-regulated expression of BSP protein continued from7d to14d, while14d later expression of Runx2protein became relatively lower than7d.Conclusion LIPUS could accelerate the proliferation of ADSCs and promote the osteogenic differentiation of ADSCs. Construction of β-catenin shRNA lentiviral vector and measurement of the silencing effect of β-catenin gene expressionObjective To construct lentiviral vector, silence β-catenin gene of rat ADSCs, provide a experimental basis for the function analysis of canonical Wnt signaling pathway in the osteogenic differentiation of rat ADSCs stimulated by LIPUS.Methods1. Three kinds of targeting sequence to silence the β-catenin gene of rat ADSCs were designed, three kinds of pCsilencerTM U6/Neo/GFP/RNAi plasmid vector were respectively constructed, then transfected into rat ADSCs and the most effective shRNA targeting sequence was chosen.2. β-catenin shRNA lentiviral vector was constructed.3. The lentiviral vector was effectively transfected into rat ADSCs. The silencing effect of β-catenin gene was measured after transfected.Results1. An effective shRNA sequence that silence β-catenin gene was chosen.2. β-catenin gene was effectively silenced by lentiviral vector transfection into rat ADSCs.Conclusion A proper method was found to stably silence β-catenin gene of rat ADSCs. The role of canonical Wnt signaling in osteogenic differentiation of rat ADSCs stimulated by LIPUS could be analysed based on it.Part IV The role of canonical Wnt signaling pathway in osteogenic differentiation of rat ADSCs induced by LIPUSObjective To explore the role of canonical Wnt/β-catenin in osteogenic differentiation of rat ADSCs induced by LIPUS.Methods1. Osteogenic differentiation related gene expressions (Runx2, ALP, BSP, Coll Ⅰ) of wild ADSCs group, Untransfected LIPUS stimulated group, Transfected LIPUS stimulated group were respectively measured by real-time PCR.2. Osteogenic differentiation related proteins (Runx2, BSP) of wild ADSCs group, Untransfected LIPUS stimulated group, Transfected LIPUS stimulated group were measured by Western blot.Results1. All the gene expressions of wild ADSCs unstimulated by LIPUS were consistent.2. Runx2gene expression of the untransfected LIPUS stimulated group was elevated at3d,5d and7d; Runx2gene expression of the transfected LIPUS stimulated group was also elevated at3d,5d and7d but with relatively smaller amplitude.3. ALP gene expression of the untransfected LIPUS stimulated group was elevated at3d,5d and7d; ALP gene expression of the transfected LIPUS stimulated group was also elevated at3d,5d and7d with the same amplitude.4. BSP gene expression of the untransfected LIPUS stimulated group was elevated at5d and7d; BSP gene expression of the transfected LIPUS stimulated group was also elevated at5d and7d but with relatively smaller amplitude.5. The expressions of Type Ⅰ Collagen gene expression in the three groups were stable.6. Runx2protein expression of the untransfected LIPUS stimulated group and the transfected LIPUS stimulated group was up regulated at7d and14d. But the amplitude at14d was smaller than that at7d. The elevated amplitude of Runx2protein in the transfected LIPUS stimulated group was smaller than that of the untransfected LIPUS stimulated group at7d.7. BSP protein expression was up-regulated in the untransfected LIPUS stimulated group at7d and14d. But the BSP protein expression of the transfected LIPUS stimulated group was elevated only at14d.Conclusion The canonical Wnt signaling pathway is blocked when (3-catenin gene of rat ADSCs is effectively silenced, which could partly inhibit the osteogenic differentiation effect of LIPUS on rat ADSCs. Canonical Wnt signaling pathway only play a limited role in the osteogenic differentiation of rat ADSCs stimulated by LIPUS.