| ATP is well known for its role as an intracellular energy source. Since 1980s, there is increasing awareness of its role as an extracellular messenger molecule and trophic factor. The role of ATP as a neurotransmitter or cotransmitter is also well established in the peripheral and central nervous systems. In addition, ATP can improve the regeneration of nerve fibre, delay the atrophy of the denervated skeletal muscles, regulate cell proliferation and differentiation and be closely relevant to cell apoptosis. Muscle-derived stem cells are a new stem cell found in skeletal muscles, and express similar surface antigens, as observed in hematopoietic stem cells, Sca-1 and CD34. MDSC can differentiate to variant tissues and posses the strong power in self-renewal. In the study of cell therapy and gene engineering MDSC is better than skeletal muscle satellite cell. We had estimated that region injecting ATP can delay atrophy of the denervated skeletal muscles, but the mechanisms are still unclear. So we designed the research and carried out it.1. Culture, identification of biological characteristics of muscle-derived stem cells from mouse skeletal muscle: we digested skeletal muscle tissues from new born mice using the dual-enzyme (collagenase XI and trypsin) digestion method, purified the cells by preplate technique and successfully cultivated muscle-derived stem cells in vitro. Immunohisochemistry staining showed that the muscle-derived stem cells expressed Sca-1 and CD34. Then the muscle-derived stem cells were cultivated and passaged in DMEM including 20% FBS. We observed their biological characteristic. We found that in low-serum media muscle-derived stem cells spontaneously differentiated to myoblast and fused into myotubes. So we conclude that mouse skeletal muscle tissues include the muscle-derived stem cells which in vitro can be passaged, cultivated, proliferate, differentiate and fuse into new myotubes, which is help to repair the injured muscles.2. Effects of ATP on the proliferation of muscle-derived stem cells in vitro: We passaged the muscle-derived stem cells in order to achieve enough cells. Then administrating ATP to the DMEM, we observed the effects of ATP on the muscle-derived stem cells by MTT method, flowcytometer and RT-PCR. The results showed that after administration of ATP, cell number evidently increased at the third day, S% and PrI increased, G1% decreased and the time of cell cycle, S and G1 phases shortened. Muscle-derived stem cells express ATP receptor (P2Y1). ATP can stimulate the expression of ATP receptor. So we think that ATP improves the proliferation of the muscle-derived stem cells in high-serum media through activating the P2Y1 receptor.3. Inhibition of ATP on TNF-αinduced apoptosis of muscle-derived stem cells: After the apoptosis of MDSC induced by TNF-α,we observed the effects of ATP on apoptosis. Apoptosis ratio of MDSC and the expressions of Caspase-3 and Caspase-8 were measured by Flowcytometer. The results showed that TNF-αcould induce the apoptosis of MDSC, while ATP could protect MDSC from the apoptosis induced by TNF-αand increase the ratio of Caspase-3~+ and Caspase-8~+ cells. So we think that ATP inhabits the expression of Caspase-3 and Caspase-8 of MDSC, which contribute to prevent the apoptosis of MDSC.4. Effects of Local ATP Injection on mouse muscle-derived stem cells of denervated skeletal muscle in vivo: We made the models of injury of sciatic nerve in mice and injected ATP into gastrocnemius as the pre-method. The proliferation of MDSC was detected post-operation. The results showed that the numbers of MDSC increased, the expression of Sca-1 in skeletal muscle tissues became stronger and the ratio of Sca-1+ cells also increased, after the injection of ATP in the normal group and denervated group. And the changes lasted over two weeks and were the most evident at the 7th day post-operation. So we think that ATP prevents the atrophy of denervated skeletal muscles, which attribute to improve the proliferation of muscle-derived stem cells.In the summary, we made use of cell isolation and culture technique, the model of nerve injury, immunohisochemistry, Flowcytometer and molecular biology technique to research the mechanisms of ATP preventing the denervated skeletal muscles from atrophy. We found that ATP can activate the P2Y1 receptor, enhance the synthesis of proteins and nucleic acid and shorten the time of cell cycle, which lead to faster proliferation of muscle-derived stem cells. In addition, ATP protects the muscle-derived stem cell from apoptosis due to denervation of muscle.
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