| Objective:Human umbilical cord mesenchymal stem cells(h UCMSCs), as a kind of more primitive stem cell, has strong capacity of repair and reconstruction of various types of damage tissue and organs. Therefore, it has become one of the hot spots in the field of stem cell research. but the biological characteristics will be influenced by the environment in the body. thereby affecting the repair of tissue and organ function. So, The focus of this study is how to make it more adaptable to the body environment, to play a better role in tissue repair. the activation of the RAS system in damage tissue resulted in angiotensin-?(Ang-?) increased, affecting the biological activity and function of endogenous and exogenous MSCs. Studies have shown that high concentration of Ang-? can induce MSCs and human umbilical vein endothelial cells(HUVEC) injury or apoptosis, even death of a variety of cells. However, the low concentration of Ang-? pretreatment MSCs, it can significantly improve their ability to withstand hypoxia, promote the secretion of a variety of cytokines.The content of vascular endothelial growth factor(vascular endothelial growth factor, VEGF) is higher and its treatment effect is the key factor In a paracrine cytokines. activate downstream signaling by binding to specific receptor(VEGFR2) and plays an important role in anti-apoptosis, promoting cell proliferation and angiogenesis, and repair and reconstruction of all kinds of damage of tissues and organs. on the basis of previous experiments and literature review, this study intends to use of appropriate concentration of Ang II pretreatment of h UCMSCs cultured in vitro. then transplant Ang-?-MSCs on skin defect animal models, to observe the efficacy of wound repair and reconstruction and in-depth study Ang-?-MSCs promoting wound healing mechanism. Further study on the mechanism of Ang-?-MSCs promoting wound healing to provide a basis for the feasibility study of stem cells for clinical treatment of multiple wounds.Method:(1)Effects of Ang-? on the biological characteristics of h UCMSCs: The experiment was divided into 4 groups. 0ng/m L Ang-?(A group), 100ng/m L Ang-?(B group), 500ng/m L Ang-?(C group)and 1000ng/m L Ang-?(D group). The cell morphology and density were observed by inverted microscope. The cell proliferation activity was measured by CCK8, The apoptosis was assessed by AO-EB staining. The cell cycle(S period) and apoptosis of h UCMSCs in four groups were tested using flow cytometry after PI staining. The levels of VEGF, b FGF, and HGF in each group were detected by ELISA.(2)Preliminary exploration of Human Umbilical Cord Mesenchymal Stem Cells pretreated with Angiotensin-?(Ang-?-MSCs) Promoting Wound Healing: Male pig with an average weight of 18 to 20 kg were used in this study. Three 5cm× 5cm full-thickness excisional skin wounds were created on each side of the midline. The skin wounds were divided randomly into the following groups: 1 m L DMEM injected into the wound at nine injection as a negative control, MSCs(treatment with 1 × 107 h UC-MSCs in 1m L DMEM injected into the wound at nine injection), Ang-?-MSCs(treatment with 1 ×107 Ang-?-MSCin 1 m L DMEM injected into the wound at nine injection). The rate of wound healing was evaluated by Image Pro Plus. The overall structure of the wound, the number of micro vessel, the expression of collagen and the expression of CK19 were detected by Immunohistochemical staining.(3)Effect of Ang-?-CM on the proliferation, apoptosis and angiogenesis of HUVEC: The experiment was divided into 3 groups. HUVEC were respectively treated with Conditioned medium group(CM), MSCs Condition Medium(MSCs-CM) and Conditional Medium from MSCs pretreated with 100ng/m L Ang-?(Ang-?-CM) to study the effect of Ang-?-CM on the proliferation and apoptosis of HUVEC. HUVEC damage induced by Ang-? 1000ng/m L was used to prepare the model of apoptosis.The cell morphology were observed by inverted microscope. The cell proliferation activity was measured by CCK8, The apoptosis was assessed by AO-EB staining, Hoechst staining and flow cytometry after PI staining. the level of Caspase-3, bcl-2 in HUVEC were investigated by western blot. Tube formation was examined by tube formation assay. On the basis of effect of Ang-?-CM on the proliferation and apoptosis of HUVEC, Further study on the protective mechanism of VEGF secreted by h UCMSCs on HUVEC. This experiment is divided into the following 6 groups: si RNA-Control + CM, si RNA-Control + MSCs-CM, si RNA-Control + Ang-?-CM, si VEGFR2+ CM, si VEGFR2 + MSCs-CM, si VEGFR2+Ang-?-CM. the apoptosis of HUVEC were tested using flow cytometry after PI staining. the level of Caspase-3, bcl-2 in HUVEC were investigated by western blot. Tube formation was examined by tube formation assay.Result:(1)Effects of Ang-? on the biological characteristics of h UCMSCs: The morphology and structure of h UCMSCs in 500 ng/m L Ang-? and 1000 ng/m L Ang-? were changed, the number of apoptotic cells was more, the proliferation activity was lower, and the level of cytokines in supernatant was lower compared with 0 ng/m L Ang-?. the group of 1000 ng/m L Ang-? were the most significant. However, the morphology and structure of h UCMSCs in 100 ng/m L Ang-? were normal, no apoptotic cells or dead cells were found, and the proliferation activity and secretion of the cytokine in 100ng/m L group were significantly higher than those of in the 0 ng/m L Ang-?, 500 ng/m L Ang-? and 1000 ng/m L Ang-?. Therefore, we choose 100 ng/m L Ang-? to pretreat of h UCMSCs.(2) Preliminary exploration of Human Umbilical Cord Mesenchymal Stem Cells pretreated with Angiotensin-?(Ang-?-MSCs) Promoting Wound Healing: In the Control group, MSCs group and Ang-?-MSCs group, the wound healing effect of Control group was the worst, Ang-?-MSCs group is the best. The healing time of the wounds in the three groups were: 55±3d, 47±3d and 40±3d. The healing time of Ang-?-MSCs group was the shortest. the healing rates of the three groups at 35 d respectively was: 63%, 75% and 88%,The wound healing rate of Ang-?-MSCs group was the highest, The structure of wound tissue of Ang-?-MSCs group was more complete, the quantity of the new micro vessel was more, the collagen arrangement was more regular and the degree of epithelium was better.(3)Effect of Ang-?-CM on the proliferation, apoptosis and angiogenesis of HUVEC: The morphology and structure of HUVEC in Ang-?-CM group and MSCs-CM group were better compared with CM group.The apoptotic cells or death cells in Ang-?-CM group was at least, angiogenesis and the proliferative activity of the cells of Ang-?-CM group was the best, The expression of anti-apoptotic protein Bcl-2 in the two groups was higher than that in the CM group, and the expression level of pro-apoptotic protein Caspase-3 was lower than that in the CM group, Ang-?-CM group was more significant. Further use of si RNA interference VEGFR2 in HUVEC, the results show that: Compared with si RNA-Control group, the expression of Bcl-2 In the si VEGFR2 group was decreased, and the expression of Caspase-3 was increased. In addition, the number of apoptotic cells in the si VEGFR2 group increased significantly than that in si RNA-Control group, while the number of angiogenesis is significantly reduced. Therefore, VEGF, VEGFR2 and expression of Caspase-3 and Bcl-2 signal is the potential mechanism of HUCMSCs promotes endothelial cell proliferation, anti-apoptosis of endothelial cells and promoting angiogenesis in endothelial cells.Conclusion:Suitable concentration of 100ng/m L Ang-? treatment can promote h UCMSCs proliferation, reduce apoptosis, secreted high levels of VEGF which combined with VEGFR2 in HUVEC, restrain the expression of downstream pro-apoptotic protein Caspase-3 and increased the expression of anti-apoptotic protein Bc-2. Play the role of promoting endothelial cell survival, proliferation and anti-apoptosis of endothelial cells, significantly promoting the wound healing of the whole skin defect of pig skin. |
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