Experiment Research On Preparation Of RT3-HG And Its Effect On Wound Healing In Diabetic Mice

【Background】Diabetic skin ulcer is one of the most common and difficult to treat chronic wounds.Compared with normal skin wound healing,diabetic skin ulcers have their own characteristics,including high glucose microenvironment,peripheral neuropathy,excessive local inflammation,migration and proliferation of vascular endothelial cells,and weakening of tube formation.Among them,the main reason for the difficulty of diabetic wound is the angiogenesis of diabetic wound.Hypoxia inducible factor-1(HIF-1)is one of the classical cytokines regulating wound healing α And HIF-1 β,The most critical one is HIF-1 α.Usually,severe ischemia caused by wound defect can activate HIF-1 α,This will further induce related cells to express vascular endothelial growth factor(VEGF),so as to enhance angiogenesis.However,the expression of HIF-1 and VEGF in the skin of diabetic patients decreased,resulting in decreased blood vessel density,poor blood circulation and impaired wound healing.In normal skin tissue,collagen mainly exists in the form of type I and Ⅲ collagen fibers,maintaining the normal tissue structure of the skin.Collagen in skin extracellular matrix is the cytoskeleton and plays an important role in the migration,proliferation and differentiation of wound healing related cells.Collagen,which can degrade rapidly,has no cytotoxicity and is easy to shape,is an excellent choice for wound covering.Additional type Ⅲ collagen supplementation can enhance HIF-1 in chronic wounds α At the same time,it increases the density of neovascularization in wound tissue.In this study,we first transfected human Ⅲ type collagen gene into yeast,and prepared high purity recombinant human type Ⅲ collagen with low cost,high productivity and no risk of disease.Then,we prepared recombinant human Ⅲ collagen sodium alginate hydrogel(Recombinant Human Collagen Type-3 Hydrogel),and detected its physicochemical properties and biological safety by mixing with sodium alginate.A recombinant human Ⅲ collagen type 10% alginate hydrogel was used as a covering to treat the wound of diabetic mice,and to observe the healing speed and the quality of skin repair.Finally,extract the extract and detect the effect of high glucose and hypoxia on the healing of diabetic ulcer healing cells and the expression of HIF-1α,The effect of VEGF and its possible mechanism.【Objective】1.Prepare different concentrations of recombinant human collagen Ⅲ sodium alginate hydrogel and detect its physicochemical properties and biological safety.2.To evaluate the effect of RT3-HG extract on the state and function of human umbilical vein endothelial cells(HUVEC)in high glucose environment and explore its possible mechanism;3.To evaluate the effectiveness of recombinant human RT3-HG in wound healing of diabetic mice and explore its mechanism.【methods and results】1.Preparation of recombinant human collagen type Ⅲ sodium alginate hydrogel and its physicochemical properties and biosafety.Methods: Different proportion of recombinant human type Ⅲ collagen,sodium alginate,double distilled water,glucose and other chemical materials were mixed with a variety of amino acids in proportion and cross-linked with different concentrations of RT3-HG.The cobalt 60 irradiation was used to sterilize and observe the appearance of the hydrogel and observe the microscopic structure of the hydrogel and take photographs.Finally,it was packed in 5 ml and placed at low temperature at 4 degrees Celsius.Different concentrations of recombinant human collagen type Ⅲ alginate hydrogel were implanted into the subcutaneous tissue of rats,and fifth,tenth,fourteenth days later,they were sacrificed and the skin tissue sections were stained with HE and immunohistochemical staining.Skin fibroblasts and immortalized keratinocytes were co cultured with RT3-HG,and the cell state was observed.Results: RT3-HG with a concentration of 10% to 30% was successfully prepared;The hydrogel is a white transparent viscous colloid,which is porous and spongy under electron microscope;The pore size range was 10-30 microns.The experiment of subcutaneous implantation in rats showed that in the skin tissue of rats,10% of the recombinant human Ⅲ collagen sodium alginate hydrogel degraded 25% in fifth days.80% on the 10 th day and 100% on the 14 th day;Immunohistochemical staining showed that 10% of the hydrogel had slight inflammatory reaction in the skin tissue,and IL-10,TNF-α,CD68 immunohistochemistry showed a continuous decrease.HSF and Ha Ca T grow normally on RT3-HG and can be cultured for more than 8 weeks.2.To evaluate the effect of recombinant human collagen type Ⅲ alginate hydrogel on HUVEC in high glucose and hypoxia environment and explore its mechanism.Methods: The effect of RT3-HG extract on the state of HUVEC in high glucose environment was detected by cell survival and death double staining method;CCK-8method was used to evaluate the effect of RT3-HG extract on the proliferation of HUVEC in high glucose environment;Cell scratch test was used to evaluate the effect of RT3-HG extract on HUVEC migration in high glucose environment;The tube forming experiment was used to evaluate the effect of RT3-HG extract on the tube forming ability of HUVEC in high glucose environment.The protein and RNA expressed after HUVEC was stimulated by RT3-HG for 48 hours were extracted,and HIF-1 was detected by immunoblotting and RT-PCR α And VEGF expression.Results: RT3-HG extract could not only improve the cytotoxicity of high glucose on HUVEC,but also enhance the migration,proliferation and tube forming ability of HUVEC.At the same time,RT3-HG extract can stimulate HUVEC to enhance the expression of HIF-1 α And VEGF.After the HIF-1 gene of HUVEC was silenced,the enhancement of HUVEC ability by RT3-HG extract was also inhibited.3.To evaluate the effectiveness of recombinant human collagen type Ⅲ alginate hydrogel on diabetic mice ulcers and explore the possible mechanism.Methods: The skin ulcer model of diabetic mice was established.The wound was covered with 10% heavy RT3-HG and sodium alginate hydrogel.The wound healing rate was calculated and wound healing time was counted.The effect of recombinant human collagen type Ⅲ hydrogel on wound healing in diabetic mice was studied.The mice were killed on days 3,7,10 and 14,and the wound tissues were stained with he,Masson,Sirius red picric acid and CD31.The protein and RNA of the wound tissues were extracted,and the wound HIF-1 was detected by immunoblotting and RT-PCR α And VEGF expression.Results: The wound observation showed that the wound surface of group RT3-HG was significantly less than that of hydrogel group seventh days after operation.On the fourteenth day after operation,the wounds healed completely in group RT3-HG,while there was a small amount of residual wounds in the hydrogel group.On the seventh day,HE and Masson staining showed that the collagen content in the RT3-HG group was significantly higher than that in the hydrogel group.Sirius red picric acid staining showed that Ⅲ collagen increased significantly in RT3-HG group compared with hydrogel group.Cd-31 staining showed that the wound neovascularization density in the collagen group was significantly higher than that in the control group.The expression of HIF-1/VEGF in wound tissue of group RT3-HG was significantly higher than that of hydrogel group.【Conclusion】1.We successfully prepared recombinant human Ⅲ collagen sodium alginate hydrogel with a content of 10% to 30%,showing a porous microstructure,strong absorption and percolation capacity,and high biosafety.Finally,10% concentration of recombinant human collagen Ⅲ sodium alginate hydrogel was selected for the following experiments.2.In high glucose hypoxia environment,RT3-HG extract can improve the toxic effect of high glucose hypoxia environment on HUVEC cells;At the same time,it can also promote the migration,proliferation and tube forming ability of HUVEC cells.The mechanism may be through the enhancement of HIF-1 α And the expression of VEGF.3.Recombinant human collagen type Ⅲ alginate hydrogel and sodium alginate hydrogel were used as cover to repair the wound of diabetic mice.Compared with the control group,the local angiogenesis of the collagen group increased significantly,while the local Ⅲ collagen content increased,thus promoting the healing of diabetic ulcer wounds.It was the first time to prove the feasibility of recombinant human Ⅲ collagen in the treatment of diabetic wounds.To sum up,the recombinant human collagen type Ⅲ alginate hydrogel was successfully prepared and applied to the treatment of diabetic ulcers.Good results were obtained.On the basis of this,the corresponding mechanism was preliminarily explored.The mechanism of recombinant human collagen Ⅲ sodium alginate hydrogel to promote wound healing in diabetes is to increase the content of Ⅲ collagen protein in wound tissue and then restore HIF-1 in diabetic wound.α The expression of HUVEC can enhance the migration,proliferation and angiogenesis of tumor cells,and increase the local angiogenesis.This provides a new way for the treatment of diabetic ulcers.