Mechanism Research Of Umbilical Cord Mesenchymal Stem Cell And Their Derived Exosomes For Cutaneous Wound Healing

Umbilical cord mesenchymal stem cells（UC-MSCs） derived from discarded umbilical cord tissue of the newborn. UC-MSCs have strong potential for self-renewal and potential for multilineage differentiation into adipocytes, osteoblasts, neural cell, chondrocytes, hepatocyte and smooth muscle cells. It is demonstrated that UC-MSCs are one of the most ideal cell on tissue engineering and regenerative medicine, because of its rich source of easily access, no ethical controversy, low immunogenicity, advantages of the proliferation and donor no trauma.However, clinical research showed that for successful stem cell therapy requires 1×109 stem cells. And in the study of stem cells therapy we need a large number of uniform sources of cells to keep the experiment results stable and contorable. So how can get such a large number of seed cells in a short time become the priority. In our study we use microcarrier and spinning bottle to culture UC-MSCs, we called it three-dimensional culture system（3D）. This culture system is simple in operation and have high efficiency can get a large number seed cells in a short time avoiding the traditional way of two-dimensional cell culture of complicated operation, easy to pollution, cell differentiation, and cell number of shortcomings. We detected the 2D and 3D cell power of proliferation through cell counting for growth curve, cell cycle and EDU flow cytometry. The results showed that the 3D culture cell have a strong power of proliferation compared to 2D culture cell. It is very important that whether the cell cultured on microcarriers keeping the characteristics of stem cells. So we use the adipogenic experiment, chondeogenic experiment and osteogenic experiment to detect the cell from 3D culture system, the results showed that the cell still retain the multipotential differentiation. The immunofluorescent staining experiment and flow cytometry experiment showed that the 3D cell still expressed the markers of VI mesenchymal stem cells. The animal experiment results showed that the cell from 2D and 3D all have the potential to enhance mouse skin wound healing. Above all we get the results that we can get sufficient quantity of UC-MSCs and the cell still have the multilineage differentiation and still express the stem cell markers, the cell from 3D culture can enhance mouse skin wound healing.We use human cells transpant to mice skin wound. There is a problem that our cells may be rejected by mice, because of the strong immune response. But in our results the rate of wound healing of UC-MSCs group is better than the control group. So we think the stem cells enhance wound healing through the paracrine function. For the problem we open the study of the mechanism of the stem cell why enhance wound healing have still unknowing.Exosomes can isolate from various cells, which is vesicle packaged by lipid bilayer membrane. They contain numerous proteins and lipids, as well as nucleic acid material in the form of DNA, m RNA, micro RNA. Exosomes become the focus of research of the cell transplantation with the advantage of more stable and reservable, have a lower possibility of immune rejection. So we think that the huc-MSCs derived exosomes（huc MSC-Ex） have the key role in wound healing experiment. The huc MSC-Ex is isolated by ultracentrifugation from the huc-MSC culture medium. huc MSC-Ex is round or ovoid with the diameter of 40-100 nm under the transmission electron microscope. The western blot results showed that the huc MSC-Ex express the markers CD9 and CD63. Above all we succeed to get the huc MSC-Ex. We know the progress of wound repair is very complex, which include cell proliferation, differentiation, migration and dead cells apoptosis, etc. Abnormal healing include the wound disunion, ulceration, scar hyperplasia and keloid formation, which is closely related to the wound cells apoptosis. We build the mode of wound disunion with 1m M H₂O₂ induced the Ha Ca T cell to apoptosis. We use different density huc MSC-Ex on the apoptosis mode, the cell proliferation assay results showed that the huc MSC-Ex can promote apoptotic cell proliferate with the dose-time effect relation. The Annexin V-PI experiment result showed that huc MSC-Ex can inhibit apoptosis rate. The reactive oxygen species（ROS） result VII showed that huc MSC-Ex can decline the ROS fluorescence intensity. Above all we get the conclusions that huc MSC-Ex can promote Ha Ca T proliferate and inhibit cell to apoptosis. In order to detect the role of huc MSC-Ex on wound healing, we use the scratch assay and transwell assay to detect the power of Ha Ca T after adding the huc MSC-Ex. The results showed that Ha Ca T migration rate of adding huc MSC-Ex is higher than control group, so we get the conclusion that huc MSC-Ex can promote Ha Ca T migrate.To detect that the mechanism of huc MSC-Ex inhibit H₂O₂ induced Ha Ca T apoptosis. Firstly, we should detect which is the H₂O₂ induced apoptosis pathway, caspase dependent or caspase independent. Tunnel and Anexxin-PI results showed that the apoptosis cells increased and the apoptosis induce factor（AIF） transfer to nuclear from mitochondria after added to H₂O₂. The western blot results showed that AIF, Cytochrome C and Calpain1 are decreased in mitochondria; and the poly（ADP-ribose） polymerase-1（PARP-1）, PAR Cy PA and AIF in nuclear are increased after adding H₂O₂. When adding PARP inhibitor DPQ, the results showed that PARP-1 the nuclear AIF is lower and PAR is regulated compared with the control group, while the group of adding the caspase broad inhibitor of Ac-DEVD-CHO showed that AIF in nuclear is not decrease and PAR express regulation. So we get the conclusion that H₂O₂ induced Ha Ca T apoptosis is from the caspase independent signaling pathway. huc MSC-Ex can inhibit this apoptosis is whether from the same pathway. Western blot results showed that huc MSC-Ex group AIF translate from mitochondria to nuclear is lower than control group and PARP-1 and PAR regulation. The PKH-26 result showed the exosomes can translate in Ha Ca T cell cytoplasm after adding huc MSC-Ex 24 hours. So we get the conclusion the huc MSC-Ex can inhibit Ha Ca T apoptosis is from the caspase independent signaling pathway and huc MSC-Ex can enter into the target cell to “cell to cell” communication.In conclusion we get the aim of get a large number of huc-MSC by the microcarriers and bioreactor culture method. Meanwhile we expanded cells still express the cell stem markers and have the multipotential differentiation and promote mouse skin wound healing. After that we want to know the huc-MSC how to work on the wound healing progress. Then we isolated huc MSC-Ex and identified it meet the criteria of exosomes. Meanwhile the huc MSC-Ex can promote Ha Ca T proliferation and inhibit H₂O₂ induced cell apoptosis and the mechanism is that the huc MSC-Ex through the caspase independent pathway in action. The huc MSC-Ex inhibit AIF translate from mitochondria to nuclear and regulation the PARP-1 and PAR express. Our study provided a new theoretical basis for stem cells translation on skin wound healing. Exosomes can avoid the shortcoming and risk of stem cells, such as oncogenicity, thrombosis, prosoplasia, immune regulation uncontrolled, etc. So exosomes therapy is expected to become the prospect for clinical treatment.