Study On The Physiological Regulation Of Mechano-growth Factor In Osteoblasts

Mechano-growth factor (MGF) is originated from alternative splicing of insulin like growth factor-1 (), which is expressed in response to the mechanic stimulation. Further studies found that MGF could promote muscle hypertrophy, preserve cardiac muscle and repair the damage of nerves. Our previous studies concluded that MGF mRNA was highly expressed in the cyclic stretched osteoblasts, suggesting that MGF might be also a mechano-sensitine molecular in bone. However, it is still unclear how MGF regulates the physiological behaviors of osteoblasts. Therefore, we constructed an recombinant adenovirus vector containing MGF gene, transfected into osteoblasts, then studied the effects of MGF on the proliferation and differentiation in osteoblasts. On the other hand, in order to investigate the influence of mechanic stimulation to osteobasts, long serial analysis of gene expression (LongSAGE)was used to analyze the difference of gene expressing between cyclic stretched cells and control cells.The main studies were as follows:1. Constructed the recombinant adenovirus vector containing MGF gene.2. Cultured, purified and identified the primary Wistar rat osteoblasts.3. Investigated the effects of MGF on the proliferation and differentiation of osteoblasts when infected by the recombinant adenovirus vector.4. Investigated the receptor pathway of MGF in osteoblasts.5. Stretched osteoblasts using the cyclic-strain unit at 15% straining, 0.5 Hz for 24 h. Unstretched cells were control. RNA was extracted from the collected cells and the difference of gene expressing between cyclic stretched cells and the control through LongSAGE was analyzed.The main conclusions were as follows:1. The recombinant adenovirus vector containing MGF gene was constructed successfully. The titre of purified recombinant adenovirus was 8.5×108 pfu/ mL.2. The primary Wistar rat's osteoblasts with typical characteristics were cultured, purified and identified. Cells from the third to sixth passages were used for the future experiments.3. The recombinant adenovirus vector was transfected into the osteoblasts. MTT method was used to analyze the effect of MGF on cell proliferation. The results showed that MGF could increase cell proliferation. Analysis of ALP activity showed that MGF could inhibit ALP activity of osteoblasts significantly.4. receptor is one of the receptor pathways mediating MGF regulating physiological effects in osteoblasts. Meanwile there is other receptor pathway in cells.5. In order to study the differences of genes expressed between stretched osteoblasts and the control, we analyzed qualitatively and quantitatively with LongSAGE. We constructed two longSAGE libraries. The result of blast showed that there are 172 tags having great differeance between the two libraries, among which 100 tags in stetched cells were up-regulated and 72 tags were down-regulated.