Relationship Research Of Mammalian Target Of Rapamycin And Wound Healing

BackgroundWound healing is one of the most important problems to the surgery and the whole medical world. And the research about healing is hot spot in the clinical cure at present. Accompanied with the development of medical and molecular biology,the human knew that wound healing can be effective controlled and explored at the gene, molecular biology and signal pathway level. Until now, the large size of the injury, or by advanced age etc who have chronic wound healing always be the hard part in the clinical wound healing. So, the research about healing mechanism has very important significance to the clinical cure.Wound healing is an extremely well-regulated and complex process accompanied with many growth factors(platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, interleukin-1) and kinds of cells(fibroblast, epidermal, endothelial cell). Many proteins will be needed in the process of wound healing, and the protein translation is essential for the process. The mammalian target of rapamycin (mTOR) signaling pathway is the major pathway for the protein translation and it can be activatied by the stimulus from growth factors, amino acids, insulin. When the pathway was activatied, it promotes the release of4E-BP1from eIF4E and recruitment eIF4A, eIF4G to make up a trimer protein structure as eukaryotic translation initiation complex eIF4F. The other hand is mTOR is important to the metabolism,cell growth and angiogenesis. In the process of wound healing, many growth factors were secreted, such as GM-CSF, VEGF, FGF, TGF-β,these factors may activate the mTOR and tanslation of the protein, then to promote the growth and proliferation and wound healing. Hence, we investigate the expression and regulation mechanism,the aim of this research not only added mechanism of wound healing, but also will provide a healing target of wound.This research first is going to make normal and GM-CSF wounded moldes and observe the level of healing, then using the ELISA, western blot,qRT-PCR to detect the expression of GM-CSF in different wounded moldes, and the mTOR signal pathway associated molecule P70S6K, phosphorylated (p-) P70S6K,4EBP-1, p-4EBP-1, mTOR, p-mTOR.,and this research will provided some new measures for the clinical cure.Methods1. Setting two groups:the control and GM-CSF group.2. Observing the percentage of wound closure in the wound healing. Collectting Wound specimens at days1,3,5,7. HE stain was used to observe histopathological changes, Massion staining of collagen and α-SMA immunohistochemistry, CD31Western blotting to observe the situation of wound healing.3. ELISA(Enzyme Linked ImmunoSorbent Assay) to detect the content of GM-CSF and qRT-PCR to detect the expression of GM-CSF.4. Western blotting to detect the mTOR signal pathway associated molecule P70S6K, phosphorylated (p-) P70S6K,4EBP-1, p-4EBP-1, mTOR, p-mTOR.5. All data were used t-test to analyze.Result1. The two groups haved the close percentage (t=0.307, P>0.05) at day1, the treated group was significantly higher than control group at days3～7post wound (t=2.704-4.030,P<0.05or P<0.01).2. Compared with the control group, Histopathologic observation found that the more number of cells and granulation tissue in treated group. During3d,5d post wound, we observed the increasing the number of vessels and obvious proliferation of epithelial cell in the wound margin, accelerating granulation tissue formation and increasing the number of vessels.3. The results of Massion shows that the expression of collagen in the GM-CSF group was higher than control group at3d-7d.4. The results of immunohistochemistry shows that the expression of α-SMA in the GM-CSF group was higher than control group at3d-7d. 5. The expression of CD31in the treated group was significantly higher than control group at daysl,3,7(t=7.237-26.40, P<0.01).6. The results of ELISA and qRT-PCR showed that the content and the relative expression of GM-CSF in both groups were all peaked at the3d of post wound, the control group was (720.87±0.89)pg/ML,2.45±0.10,the treatment group was (910.50±1.33)pg/ML,2.80±0.48. The content of GM-CSF in the treated group was significantly higher than control group(t=105.743-298.971, P=0.000) at every time point. The relative expression of GM-CSF in the treated group was significantly higher than control group at days1,5,7(t=4.070-5.275, P<0.01).7. The expression of m-TOR and phosphorylated m-TOR in the treated group was significantly higher than control group at every time point (P<0.05or P<0.01).The expression of P70s6k was significantly higher than control group at days3,5,7(P<0.05or P<0.01). The expression of phosphorylated P70s6k was significantly higher than control group at days1,3,7(P<0.05or P<0.01). The expression of4E-BP1in the control group was significantly higher than treated group at daysl,3,5(P<0.05or P<0.01), but the phosphorylated4E-BP1was significantly higher than control group at days1,3,7(P<0.01).ConclutsionGM-CSF could accelerate wound healing by activating mTOR pathway accompanying phosphorylation of downstream molecule (4E-BP1 and P70S6k).