| Background and purpose:Stem cells are cells in the body that are self-proliferating,multi-directional,and have powerful paracrine functions.Adipose stem cells,as stem cells that can be obtained from adipose tissue in human body,are widely used in clinical practice because of their easy availability.Its powerful self-proliferation and paracrine ability can be applied to cell therapy for the treatment of a variety of diseases,such as refractory wounds of diabetic foot[1].However,during the in vitro culture and expansion of stem cells,the cells will age,the morphology will change,the proliferation and differentiation potential will be significantly reduced,and the cells will be more susceptible to external environmental effects and die[2].This undoubtedly greatly affects the therapeutic effect of the cells.There are many ways to improve the therapeutic potential of stem cells,such as artificial scaffold materials[3],xenobiotic biomaterials[4]and so on.These methods inevitably introduce foreign materials or add chemical agents that may be harmful to the body[5].The relatively high cost of treatment itself,but also the introduction of relatively potential risk factors,may cause damage to the body.According to previous experiments by research team members,it can be known that hyaluronic acid,which is present in the extracellular matrix,can be used for three-dimensional cell culture[6].It has very safe biological properties.The three-dimensional culture of microspheres can increase the stem cell potential of stem cells,the stem cell markers of cells are obviously up-regulated,and the cell survival rate is greatly increased[7],and the proliferation and differentiation of the paracrine function is significantly improved.At the same time,it is derived from endothelial cell colony forming cells in peripheral blood,which has a very strong self-proliferative potential.Among them,endothelial cell colony forming cells with high value-added potential have colony forming ability.It interacts with adipose stem cells.On the one hand,it can increase its stern cell potential.On the other hand,it can increase the survival of adipose stem cells[8].The purpose of this experiment is to increase the stem cell potential of adipose stem cells by reducing the stem cell potential of adipose stem cells by reducing the stem cell potential of adipose stem cells by using three-dimensional structure of hyaluronic acid three-dimensional culture after repeated passage of aging fat stem cells and endothelial cell colony forming cells,thereby increasing the disease The therapeutic effect.Experimental method:1.Obtain aging adipose stem cells after repeated passageConventional use of 3.5mm diameter multi-side liposuction for lipolysis of healthy women s abdominal fat.The obtained fat is poured into a blood bag and allowed to stand in the ice water mixture until the blood water and the upper layer of fat are clearly separated.Release the lower layer of blood and leave the upper layer of fat in a 50ml centrifuge tube to half.25 ml of phosphate buffered saline solution was added to the centrifuge tube,shaken well,and centrifuged at 1200 g for 3 minutes in a centrifuge.Discard the lower layer of liquid,retain the middle layer of fat,and remove excess oil from the upper layer.The 0.075%collagenase I solution was configured to digest the adipose tissue and placed in a 37-degree water bath shaker for 30 minutes.The collagenase I was neutralized with whole culture,and after centrifugation,the upper oil droplets and the undigested adipose tissue were removed,and the lower cell pellet was inoculated on the culture dish.Repeated culture until the sixth generation(that is,aging fat stem cells).2.Obtaining endothelial cell colony forming cells20 ml of healthy women's peripheral blood was taken,and the lymphocyte separation solution was added after mixing 1:1 phosphate solution.Centrifuge at low speed for 30 minutes.Take the middle white cloud-like cell layer,the mononuclear cells in the blood.Inoculate in a petri dish of collagen I plating until cell colonies appear.When the number of cells is about 2000,it is passaged.3.The experimental animal selected in this experiment is a nude mouse.A small cylindrical lead column is made,and a gap is left in the middle to remove the skin of the nude mouse,and the transparent tape is fixed.After anesthetizing the nude mice,they were fixed under a radioactive source and irradiated with 15 Gray.Waiting for one month to cause chronic fibrosis of the skin of nude mice,at this time cut out a 7mm full-thickness skin wound with cut out.The wound was treated with the three-dimensional cultured cells and the control group.The control group was di'vided into:sixth-generation adipose stem cells,three-dimensional cultured sixth-generation adipose stem cells,endothelial cell colony forming cells,and endothelial cell colony forming cells.4.Experimental results:Three-dimensional culture of endothelial cell colony forming cells and adipose stem cells appeared in a similar structure to the vascular structure.Trying to use the sixth generation of adipose stem cells in three-dimensional culture,there are a large number of individual cells that cannot form a spatial structure,which float in hyaluronic acid,death.As the wound treatment time went on,all the experimental and control wounds tend to heal.However,the three-dimensional culture of endothelial cell colony forming cells and adipose stem cells was the best in the treatment group,and there was no significant difference between the other three groups.The three-dimensional culture group survived better tissue and participated in the formation of vascular structures,while the simple adipose stem cell group was metabolized in the host body and only a small amount remained in the interstitium,and it was not involved in the body tissue structure.The regeneration of the tissue was also significantly inferior to that of the three-dimensional culture group. |
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