| Backgroud Recently, the research report about MSCs is more and MSCs have been wildely used to treatment of multiple human diseases. Previous studies demonstrated that, in animal models and clinical trials, applying MSCs could efficiently relieve rheumatoid arthritis and promote the regeneration of tissues. Our previous dates also showed that, MSCs efficiently promoted the angiogenesis during skin wound healing and restrained skin hyperplastic scar. Bone mesenchymal stem cells(BMMSCs) isolated from bone marrow is an acceptable MSCs and have been wildely used in clinical. Jaw of bone marrow mesenchymal stem cells(JBMMSCs) isolated from jaw bone is a kind of adult stem cells, posses the similar ability of self-renewal and multiple differentiation potential as BMMSCs. However, the main reason for the declining therapeutic effect of MSCs is that microenvironment(inflammation) affected the biological properties(paracrine) of JBMMSCs.Autophagy is a process that intracellular vesicles packaged misfolded proteins and damaged organelles such as mitochondria and endoplasmic reticulum, fused with lysosome to form autolysosome, degraded and recycled its contents, in order to realize metabolism and renew the intracellular organelles. Extracellular signal regulated kinase(ERK) is an important kinase that regulates the function of cells, and interplay with autophagy-related genes(ATGs). Once the gene of ATG5/7 were knockout, the phosphorylation of ERK would declined. Previous research reported that autophagy would promote the treatment effect of BMMSCs by regulating its functions, but the role of autophagy on the paracrine of JBMMSCs and the mechanism are not yet clearly. Therefore, in this study, we firstly compared the angiogenesis of ECs cultured with H/P-JBMMSCs and the level of autophagy in JBMMSCs isolated from normal and inflammatory jaw bone, and then studied the regulation and mechanism on the paracrine function of JBMMSCs through autophagy, providing a new idea to invert the paracrine function and deeply study the biological properties of JBMMSCs.Aim 1. Investigate the influence of inflammatory microenvironment on the osteogenesis and pro-angiogenesis in JBMMSCs; 2. Investigate the influence of inflammatory microenvironment on the level of autophagy in JBMMSCs; 3. Investigate the influence of autophagy in the angiogenesis of ECs cultured with JBMMSCs; 4. Investigate the reversion of pro-angiogenesis of JBMMSCs by activating autophagy; 5. Investigate the regulation and mechanism on the paracrine function of JBMMSCs through autophagy.Methods Part One: The differences of biological properties in JBMMSCs isolated from normal and inflamed jaw bone. First, we collected the normal jaw bone tissues from 4 patients undergoing orthognathic surgery, and the inflamed jaw bone from 3 patients diagnosed with osteomyelitis. As the method of isolating the mesenchymal stem cells from the normal jaw bone tissues, we successfully isolated the P-JBMMSCs from inflamed jaw bone. Then we tested the ability of cell proliferation, CFU-F and the osteogenic/ adipogenic differentiation potential of H-JBMMSCs and P-JBMMSCs. Further, we compared the differences of osteogenic differentiation potential by ALP staining、alizarin red staining and western blot, the tube formation of ECs by Matrigel text between H-JBMMSCs and P-JBMMSCs. Part two: The differences of autophagy level of JBMMSCs isolated from normal and inflamed jaw bone. First, we tested the expression of LC3 and Beclin-1 by western blot and RT-PCR. Second, immunofluorescence staining was used for determining the expression and distribute of LC3. Finally, we observed the ultrastructure of autophagosome by transmission electron microscopy(TEM) in H-JBMMSCs and P-JBMMSCs. Part three: To investigate whether autophagy could regulate the angiogenesis of ECs after being co-cultured with JBMMSCs. We tested the angiogenesis protein(VEGF、Ang II、PCNA) of ECs by western blot and the tube formation by Matrigel text of ECs after being co-cultured with JBMMSCs which had been treated with rapamycin or si-Beclin-1. At the same time, in order to avoid the effects of autophagy on the angiogenesis of ECs directly, we tested the angiogenesis ability of ECs after being treated with rapamycin or si-Beclin-1. Then the skin wound model was established, we photoed the general picture at 7 and 14 days after injecting JBMMSCs, observed the wound healing by HE staining and new capillaries by inverted microscope, tested the expression of CD31 by immunofluorescence staining. Part four: Rapamycin reverse the angiogenesis of ECs after being co-cultured with P-JBMMSCs. In order to clear the correlation between autophagy and the angiogenesis of ECs after being co-cultured with P-JBMMSCs, we tested the angiogenesis protein(VEGF、Ang II、PCNA) of ECs by western blot and the tube formation by Matrigel text of ECs after co-cultured with JBMMSCs which had been treated with rapamycin. Then the skin wound model was established, we photoed the general picture at 7 and 14 days after injecting JBMMSCs, then observed the wound healing by HE staining and new capillaries by inverted microscope, tested the expression of CD31 by immunofluorescence staining. Part five: Autophagy promote the paracrine of VEGF in JBMMSCs through regulating ERK pathway. First, we screened out the vascular-associated factor by RT-PCR and verified the the effect of this factor on the angiogenesis of ECs. Then we texted the binding of protein ERK and LC3 by Co-IP, the protein expression of Raf-MEK-ERK pathway by western blot. In order to clear the VEGF was secreted through the Raf-MEK-ERK pathway, we text the expression of VEGF after inhibiting ERK pathway with U0126.Result Part one: The comparison of biological properties in JBMMSCs isolated from normal and inflamed jaw bone. 1. We successfully isolated JBMMSCs from normal and inflamed jaw bone tissues and verified the ability of cell proliferation, CFU-F and the osteogenic/adipogenic differentiation potential of H-JBMMSCs and P-JBMMSCs; 2. Compared with H-JBMMSCs, the potential of osteogenic differentiation in P-JBMMSCs was significantly decreased; 3. After co-culturing with H-JBMMSCs and P-JBMMSCs, the ability of angiogenesis of ECs was lower in P-JBMMSCs than H-JBMMSCs. Part two: The low level of autophagy in JBMMSCs isolated from inflamed jaw bone. 1. Our dates showed that the expression level of LC3 and Beclin-1 was declined in P-JBMMSCs by western blot and RT-PCR. 2. Immunofluorescence staining date revealed that the expression of LC3 was weakened in P-JBMMSCs than H-JBMMSCs. 3. We could not found obvious autophagosome in P-JBMMSCs by transmission electron microscopy(TEM). Part three: Autophagy up-regulate the angiogenesis of ECs after co-cultured with JBMMSCs. 1. The angiogenesis ability of ECs had no significant change after being treated with rapamycin or si-Beclin-1 directly.2. The effect of wound heaing using the method of subcutaneous injection was much more better than intravenous injection. 3. The angiogenesis ability of ECs was changed after co-cultured with JBMMSCs which had been treated with rapamycin or si-Beclin-1. 4. There was no influence on apotosis of JBMMSCs when had been treated with rapamycin or si-Beclin-1. 5. In vivo results showed that, JBMMSCs treated with rapamycin would promote the vascularization of the skin and accelerate wound healing. Part four: Rapamycin reverse the angiogenesis of ECs after being co-cultured with P-JBMMSCs. 1. Rapamycin had no effect on the wound healing through applying locally. 2. Rapamycin could reverse the angiogenesis of ECs after being co-cultured with P-JBMMSCs. 3. After activating the autophagy, P-JBMMSCs could promote the vascularization of the skin and accelerate wound healing. Part five: Autophagy promote the paracrine of VEGF in JBMMSCs through regulating ERK pathway. 1. We successfully screened out the vascular-associated factor VEGF by RT-PCR. 2. In vivo results showed that, the expression of VEGF in rapamycin group was increased. 3. VEGF promote the angiogenesis in skin wound healing. 4. Autophagy promote the paracrine of VEGF in JBMMSCs through regulating Raf-MEK-ERK pathway.Conclusion 1. In this study, we successfully isolated JBMMSCs from normal and inflamed jaw bone tissues and verified the ability of cell proliferation, CFU-F and the potential of osteogenic/adipogenic differentiation in H-JBMMSCs and P-JBMMSCs. Further, we found that the potential of osteogenic differentiation in P-JBMMSCs and the angiogenesis of ECs co-cultured with P-JBMMSCs were reduced in inflamed microenvironments. 2. Autophagy affected the angiogenesis of ECs co-cultured with JBMMSCs isolated from inflamed jaw bone tissues. 3. Autophagy up-regulate the angiogenesis of ECs after co-cultured with JBMMSCs. The angiogenesis ability of ECs was increased after co-cultured with JBMMSCs which had been treated with rapamycin, and declined after co-cultured with JBMMSCs which had been treated with si-Beclin-1. 4. Rapamycin reverse the angiogenesis of ECs after being co-cultured with P-JBMMSCs. 5. Autophagy promote the paracrine of VEGF in JBMMSCs through regulating Raf-MEK-ERK pathway, and VEGF promote the angiogenesis of ECs. In summary, this study demonstrated that, the ability of angiogenesis in ECs was decreased after being co-cultured with JBMMSCs in inflammatory microenvironment, and this ability was reversed by activating the function of paracrine in JBMMSCs through autophagy. In this study, we not only clear the regulation function of JBMMSCs in inflammatory microenvironment, but also suggest autophagy play an important role in regulating function of paracrine in JBMMSCs, providing a new theoretical and experimental basis for better and more further research of JBMMSCs. |
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