| According to the different parts of the skin, as well as different causes in different injury, wound can be divided into many types, but the healing process of skin wounds is very similar, the complex and regular repair process, including the inflammatory response, granulation tissue formationstage, tissue regeneration, and the formation of scar tissue.Mild wound limited to the skin epidermis heal throgh epithelial regeneration, but fracture associated with the skin and subcutaneous tissue of the heavier, larger wounds, as well as associated with infections, and a variety of systemic diseases refractory wound, usually require manual intervention, such as the use of a variety of cytokines, the use of a variety of excipients, and the skin patch and skin graft, etc. And skin appendages (hair follicles, sweat glands and sebaceous glands) as being completely destroyed, can not be completely regenerated, and scar repair. With the development of the basic research and biological materials technology advances, more and more the role of cytokines mechanism can be described and used in the clinical application, and biological material is applied at the site of the wound to prevent infection and the loss of body fluids. Activin B is the one of TGFβ superfamily members, our previous study found that Activin B adjust keratinocyte cell actin reorganization to promote its migration, In this study we will explore what the role og Activin B in the process of wound healing, and to further explore the signal mechanism; Temperature sensitive molecular modification of the hydrogel, which may be rendered according to the temperature change in a different state, below 33℃ is the liquid state, higher than 33℃ is jelly. According to this feature, it can meet demand of different morphological wounds; this study will explore its role in wound healing, and to provide a basis for its further application in wound healing.Chapter One Activin B Promotes Epithelial Wound Healing In Vivo through RhoA-JNK Signaling PathwayBackground and PurposeSkin wound healing including the regeneration of the epidermis and dermis, one of the most important events in the epidermis regeneration process:the keratinocyte migration to the wound of epithelial and ancillary structures around the wound, series of phenotypic changes in cells migration process; the followed immediately occurrence thing is cell proliferation activities, keratinocyte cells induced by cytokines or physical chemical signal after the traumatic event are through migration, proliferation process and then the formation of the newborn epidermis is the skin re-epithelialization.Two core issues of wounds in the skin areas:one is the speed of wound healing; the other one is wound healing quality problems. Rapid healing of wounds can reduce wound infection and reduce the formation of hypertrophic scars, so to explore how to promote the proliferation and migration of keratinocytes around wound is the crux of the problem during wound healing. Our group early findings that activin B promotes keratinocytes migration through adjust actin reorganization of keratinocyte in vitro, and this phenomenon relies on the RhoA-JNK and MEKK1-p38 signaling pathway, which provides the basis for us to explore how to enhance the level of wound healing rate in in vivo. Activin B is the one of transforming growth factor-β superfamily members, and plays a biological effect thereon the activin B receptors binding, follistatin is activin antagonists of solubleprotein, follistatin can be combined with Activin B, and inhibit the biological effects of Activin B by inhibiting the combination of activin B to its receptor. RhoA is the one of the members of the Rho family, MEKK1, JNK, p38 signal belongs to the mitogen-activated protein kinase (MAPK) family members, is an important signal system response mediated cell biology, and transfer signal using highly conserved grade kinase cascade. Combined with the preliminary research we asked the following question:Activin B can promote keratinocyte migration accelerated wound healing in vivo? RhoA-JNK and p38 signal whether or not to participate in skin wound healing process?Research MethodsWound model:Kunming (KM) mice,0.5cm× 0.5cm square incision on back left. Group:PBS group, Activin B group, Plenti6/v5-EGFP group, Plenti6/v5-EGFP-RhoAL63 group, Plenti6/v5-RhoAN19 group, Y27632 group, Plenti6/v5-EGFP+ Activin B group, Plenti6/v5-EGFP-RhoAL63+Activin B group, Plenti6/v5-RhoAN19+Activin B group, Activin B+Y27632 group, Activin B+ SP600125 group, SP600125 group, Plenti6/v5-EGFP-RhoAL63+SP600125+ Activin B group, Plenti6/v5-EGFP+SP600125+Activin B group, Activin B+ Follistatin group, Follistatin group.Detection indicators:(1) organizational form:after three days, five days, seven days recording the wound area to calculate the rate of wound healing; using the H&E staining to observe the wound re-epithelialization; ultrastructure of wound healing changes observed by scanning electron microscopy and transmission electron microscopy; (2) signaling mechanisms:using immunning histochemical to detect BrdU-positive keratinocyte proliferation around the wound; the phosphorylation of the protein kinase:JNK, Rock, ERK, p38, p-JNK, p-ERK, p-p38 and p-cJun were detected by Western Blotting technology and immunohistochemicalion.The resultsThe rate of wound healing:The results showed that in vivo Activin B significantly promote wound re-epithelialization, improve the rate of wound healing compared with the PBS group (F=359.810, P <0.001);Tissue morphology:Activin B promotes the migrate of keratinocytes around wound to the wound site, on day three and five epithelial keratinocytes cell layers around the wound in Activin B group were more than the PBS group, on day six re-epithelializationhas been completed, and found that the regeneration of the hair follicle, but on day six PBS group is still not yet complete re-epithelialization. Scanning electron microscopy and transmission electron microscopy found:on day three there are re-epithelialization of new epithelial cells and a large number of keratinocytes crawling along collagen in Activin B group. On day three there are re-epithelialization of new epithelial cells in PBS group, but thr number of keratinocytes crawling along collagen was less than the number in Activin B group. On day five the basal cells of the PBS group was connected more closely than Activin B group, this suggesting that the basal cell of Activin B group were more conducive to migration. These results suggest that Activin B in vivo promote keratinocyte migration.Signaling mechanisms:immunohistochemical detection found that Activin B promotes the keratinocytes proliferation around wound, Plenti6/v5-EGFP-RhoA L63 can further improve the rate of wound healing; Activin B promote phosphorylation of JNK and cJun using immunohistochemistry and WB, the above results suggest that RhoA, JNK and cJun involved the process of Activin B-induced wound healing. Further studies showed that Plenti6/v5-EGFP-RhoAN19, Y27632, and SP600125 well Follistatin can inhibit Activin B-induced the rapid re-epithelialization and the keratinocytes proliferation around wound, the above findings verify that Activin B promote skin wound healing through RhoA-Rock-JNK-cJun signaling pathwayConclusionsActivin B promotes the keratinocytes proliferation around wound and epithelial wound healing in vivo through RhoA-Rock-JNK-cJun signaling pathwayChapter Two The temperature-sensitive hydrogel promote skin wound healingBackground and purposeThe skin is the largest organof body’s, the 5%-15% of total weight, the 1.5-2 square meters of total area, and its primary function is to protect body isolated from the external environment as a protective barrier. At the same time, the skin damage caused due to trauma, burns, infections, tumors, and metabolic diseases are very common in clinical. The key for the treatment of skin defect is to be wound closed as soon as possible order to reduce the incidence of complications of body, and the interal environment disorder, as well as microbial invasion. It is difficult for larger wound to be wound closed as soon as possible and to achieve rapid healing through autologous biological activities, so biological scaffolds and biological scaffolds complex cytokine and/or seed cells was applied.The more application of material are various preformed scaffold and a hydrogel, and hydrogel has many advantages with respect to the preformed scaffold:Initial sol/ cell suspension may be filled with the arbitrary shape of the defect, and can be treated with various drugs mixed, without surgical implantation. Therefore, the in situ formation gel is used in a wide range of applications in tissue engineering. The hydrogel material can be divided into different types based on the different points of the modifying molecule; temperature-sensitive hydrogel is temperature sensitive hydrogel, which haves the lowest critical solution temperature (LCST), a sudden contraction in the LCST volume will be near orexpansion that occurred sol phase to volume contraction phase changes. In this study we use the specially modified temperature-sensitive hydrogel (Xing Mengqiu Professor, University of Manitoba, Canada) to explore the role of the modified temperature-sensitive hydrogel in wound healing process. The major biological event in the process of wound healing: inflammation reaction (mainly macrophages) and granulation tissue (angiogenesis, fibroblast proliferation), the proliferation and migration of keratinocytes, shut contraction and collagen production and degradation. In this study, we will explore the role of the temperature-sensitive hydrogel in the above biological time.Research MethodsWound model:C57BL/6 mice, 1cm X 1cm square incision on back left.Group:Control group, Hydrogel-3 group, and Hydrogel-1 group.Detection indicators:(1) organizational form:on 0-7 days after wound, recording the wound area to calculate the rate of wound healing; H&E staining for wound re-epithelialization; (2) macrophage infiltration:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry to detect the expression of the macrophage marker F4/80 and statistical analysis; (3) the number of pericyte in granulation tissue: on 3 days and 5 days after wound, paraffin sections, immunohistochemistry to detect the expression of pericyte marker--a-SMA and statistical analysis; (4) the number of myofibroblasts in granulation tissue:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry to detect the expression of myofibroblasts marker--α-SMA and statistical analysis; (5) the the proliferation of keratinocytes:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry to detect the expression of keratinocytes proliferation marker-K6 and statistical analysis; (6) collagen staining:on 5 days,7days and 14 days after wound, paraffin sections Masson three-color staining to detect the expression of collagen in woud site.The results:Wound healing rate:The results showed that the temperature-sensitive hydrogel significantly increased the rate of wound healing (F=190.303, P<0.001) compared with the PBS group;The morphology observed:wound re-epithelialization was observed in the light microscope level during postoperative day 3,5 and 7 days, respectively, to take the tissue around the wound 0.5cm within. H&E staining found that 3 days after wound there are more cell layers in Hydrogel-3 and Hydrogel-1 group wound edge and keratinocytes migrate to the wound center to form the epithelium belt (re-epithelialization), but there is only the proliferation of keratinocytes and the belt is not obvious in Control group. On day 5 there are more and more cell layers in Hydrogel-3 and Hydrogel-1 group wound edge, and the belt is longer than Control group. And On day 7 Hydrogel-1 group wound has completed re-epithelialization, wound has been largely completed the re-epithelialization in Hydrogel-3 group. The results suggest that the Hydrogel promote wound contraction at the same time promote wound re-epithelialization.Detection indicators:(1) macrophage infiltration:On day 3 and 5, paraffin sections, immunohistochemistry showed that Hydrogel promote infiltration of macrophages; (2) the number of pericyte in granulation tissue:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry showed that Hydrogel promote the generation of pericyte in granulation tissue. (3) the number of myofibroblasts in granulation tissue:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry showed that the number of myofibroblasts in Hydrogel group is more than the number in Control group (F=316.230, P<0.001), suggesting the Hydrogel possible promote wound contraction by myofibroblasts; (4) the proliferation of keratinocytes:on 3 days and 5 days after wound, paraffin sections, immunohistochemistry showed that Hydrogel promote the proliferation of keratinocytes around wound. (5) collagen staining:on 5 days,7days and 14 days after wound, paraffin sections, Masson three-color staining showed that Hydrogel promote the generation and degradation of collagen.ConclusionsThe temperature-sensitive hydrogel promote rapid re-epithelialization of the wound though adjust trauma and then wound contraction. |
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