Elucidation of the effects of Dlx5 on osteogenic differentiation of mesenchymal stem cells

• Main Authors: Tien-Ching Chang, 張恬菁
• Other Authors: Yeu Su
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/28227450290374552777

碩士 === 國立陽明大學 === 生物藥學研究所 === 98 === Mesenchymal stem cells (MSCs) was first found in bone marrow by Friendenstein et al. MSCs are multipotent stem cells, MSC can differentiatie into different cell types, such as osteoblasts, chondrocytes, adipocytes and cardiomyocytes. MSCs have good ability of proliferation and potentiation of differentiation, therefore it is suitable for MSCs to apply for the materials of tissue engineering. My research focus on the regulation of transcription factors for the osteogenic differentiation of mesenchymal stem cells. Bone cells are differentiated from mesenchymal stem cells, osteoprogenitors, preosteoblasts, osteoblasts and finally mature osteocytes. Dlx5 is a transcription factor containing homeodomain. The mice lacking Dlx5 developed severe craniofacial defects and abnormal bone development, and then died after birth. Dlx5 overexpression in cells can stimulate the expression of other downstream genes of osteogenic differentiation and facilitate the progression of osteogenic differentiation. Therefore, Dlx5 plays a role as a positive regulator for osteogenic differentiation. I focus on elucidating the effects of Dlx5 on osteogenic differentiation. So far, I purify the adenoviruses containing the Dlx5 gene, and use the adenoviruses to infect the MSC-like stem cells, C3H10T1/2, and then to elucidate the effects of Dlx5 on osteogenic differentiation in C3H10T1/2. First, I use the adenoviruses containing Dlx5 to infect C3H10T1/2 alone, and then detect the expression of osteogenic differentiation marker gene alkaline phophatase (ALP). Dlx5 can stimulate the expression of ALP of C3H10T1/2 cells at 14 days post-infection. In addition, co-infection of adenoviruses containing Dlx5 and Cbfa1 in C3H10T1/2 induces more significant ALP activity than that of myc-Cbfa1 expression alone. Moreover, it also shows that the protein expression level of myc-Cbfa1 increases after co-expression of Dlx5 and myc-Cbfa1, while the exogenous myc-Cbfa1 mRNA expression level, maintain equal. Therefore, Dlx5 may stabilize myc-Cbfa1 protein, and then I used cycloheximide to inhibit the protein synthesis in order to trace the protein stability of myc-Cbfa1. Whether Dlx5 can delay the myc-Cbfa1 protein degradation still need to be confirmed. Moreover, I used MG132 to inhibit proteosome degradation to measure the protein levels degraded by this pathway. It shows that Dlx5 can indeed protect myc-Cbfa1 protein from degradation through proteosome pathway. The results suggest that Dlx5 can stimulate osteogenic differentiation by stabilizing myc-Cbfa1 protein.