脂肪干细胞 z-bo 2020-12-24 309

　　Introduction: After dermal tissue injury, abnormal extracellular matrix deposition and remodeling, especially collagen, can cause hypertrophic scars. This scar tissue usually becomes stiff due to itching, pain, and redness, causing significant appearance and function problems for the patient. There are various treatments for hypertrophic scars including resection, intralesional steroid injection, compression, laser, and interferon injection. However, none of these treatments have been proven to be effective in preventing excessive scar tissue formation and healthy dermal tissue regeneration. Therefore, the treatment of hypertrophic scars remains a challenge. Hypertrophic scars are caused by specific factors in the wound healing process, including inflammation, hyperplasia, and remodeling. The immune dysfunction of T cells and macrophages, accompanied by a wide range of inflammatory reactions, can form void filling, non-functional tissues, leading to the evolution of scars. Reactive oxygen species (ROS) is also a powerful driving factor for collagen deposition. It is a highly cytotoxic compound secreted by neutrophils and used for wound disinfection. Transforming growth factor β1 (TGF-β1) is a known dermal fibrosis booster and an important collagen stimulating factor in fibroblasts. In addition, TGF-β1 inhibits the expression of matrix metalloproteinase (MMP), leading to the accumulation of collagen fibers in the wound. Myofibroblast differentiation is another fibrosis enhancer in the process of wound healing. Myofibroblasts differentiate into fibroblasts in the injured environment in order to shrink the edges of the wound and accelerate re-epithelialization through contraction. Tension, excessive, and irregularly arranged collagen fiber bundles lead to hyperproliferative hypertrophic scars. Prolonged wound healing usually leads to hypertrophic scars. During the healing process, it is very important to ensure that a proper microvascular network is formed and developed into a permanent vascular network. Otherwise the wound closure will be damaged and hypertrophic scars will appear. In the process of wound healing, any abnormality will have a negative impact on tissue regeneration and contribute to the formation of hypertrophic scars. From a therapeutic point of view, drugs that treat abnormalities during wound healing may help treat scars. Adipose stem cell (Adipose Stem Cell) treatment is promising to prevent fibrosis by reducing inflammation and inhibit TGF-β1, which is beneficial to tissue regeneration at the wound site.

　　 Method: Rabbit ear hypertrophic scar model: 12 adult New Zealand albino rabbits (each weighing 2.5 to 3 kg) were anesthetized under aseptic conditions to prepare wounds. Use a trephine to prepare 6 round, 1 cm wounds on the exposed cartilage on the ventral surface of each ear, and carefully remove the epidermis, dermis, and perichondrium. The wound was covered with erythromycin ointment, and the secretions were washed the next day. This study excluded samples of infectious or necrotic wounds.

　　 Preparation of adipose stem cells and conditioned medium: Four-week-old New Zealand white rabbits were sacrificed, the inguinal fat pads were collected, minced, and digested with 0.075% type I collagenase at 37°C, continuously shaking for 45 minutes. Centrifuge the cell suspension at 1200×g for 10 min. Suspend the fat cells in a low-sugar DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and at 37°C containing 95% air, Cultivate in an environment with 5% CO2. Change the medium every 3 days. A six-well plate was used to seed ADSCs (about 5×104 cells/cm2). Sixteen hours after sowing, the conventional medium was replaced by DMEM medium without fetal calf serum. Finally, incubate for 48 h, collect the culture supernatant, centrifuge at 300×G for 5 minutes, and filter through a 0.22 micron filter.

　　 Identification of ADSCs: Cells were detected by flow cytometry. The third generation ADSCs were incubated with monoclonal antibodies for PE-labeled CD34, CD105, CD73, HLA-DR antibodies or FITC-labeled CD14, CD90, and CD45 antibodies for 30 minutes at room temperature. Cells stained with isotype control antibody are used as controls. The cells were washed with phosphate buffered saline (PBS), then fixed with 4% formaldehyde, and analyzed with a FACScan flow cytometer. And evaluate fat formation and differentiation according to previous methods.

　　Intralesional injection: Collect the third-generation adipose stem cells and label them with DIL. The adipose stem cells were washed twice with PBS, suspended in Dil dilution (5μL/m DMEMl medium), and incubated at 37°C for 20 minutes. After incubation, rinse twice and resuspend in low-sugar DMEM medium. Use a 29G syringe to slowly inject 0.2 ml of DMEM medium containing 4 million adipose stem cells into the center of each lesion from the edge of the wound. Similarly, 0.2 ml of ADSCs-CM was injected into the center of each lesion.

　　 Scar evaluation: Observe the external changes of the wound every week after the operation. Before operation, 2, 3, 4, and 5 weeks after the operation, the ultrasound images of the lesion were examined to record the internal changes of the scar.

　　 Histological analysis: The scar was taken 35 days after operation for histological examination. Fix them immediately at 10% formalin. Then the scar is implanted in paraffin sections. For the Scar Elevation Index (SEI), sections were stained with HE and examined under a microscope. The measurement was performed twice by a blind examiner and the average value was used. The arrangement of collagen fibers was further subjected to Masson's trichrome staining method.

　　 Tracking fat stem cells: the scar tissue is divided into two, fixed in 4% paraformaldehyde containing 10% sucrose for 12 hours at 4°C, and then fixed in a solution containing 30% sucrose and 4% paraformaldehyde for 24 hours. The specimen is then implanted in the OCT complex and stored at -80°C until use. Cut into 10um sections and rinsed with PBS 3 times. After air drying for 10 minutes, they performed DAPI (1μg/ml) nuclear staining and photographed with a fluorescence microscope.

　　Total RNA extraction and real-time quantitative polymerase chain reaction: After harvesting scar tissue, immediately freeze it with liquid nitrogen. After homogenizing the tissue, follow the kit instructions to extract RNA and perform gene expression analysis.

　　Result: ADSCs identification: In conventional culture media, adipocytes are in the form of fibroblasts and are easy to expand. They were confirmed to be positive for CD73, CD90, and CD105. It can also successfully reverse differentiate into adipocytes and bone cells.

　　 ADSCs and ADSCs-CM can also reduce scar hyperplasia: 14 days after surgery, gross examination revealed that the wound was completely re-epithelialized. In the control and untreated groups, re-epithelialization, stiffness and obvious raised scars gradually formed. Scars were significantly improved in the ADSCs and ADSCs-CM groups.

　　 Ultrasound inspection and calculation of SEI: We use ultrasound to monitor wound changes. SEI is calculated by recording the precise thickness of scar tissue from epithelium to cartilage. This is very different from the traditional method of measuring total thickness, including the underlying cartilage and tissue. Ultrasonography of trauma showed that the SEI of the treatment group was significantly different from the control group and the untreated scar group.

　　 HE staining and calculation of SEI: On the 35th day after the operation, HE staining showed that the control group and the untreated group had mild contraction scars and significantly thickened. The scars in the ADSCs and ADSCs-CM groups were flat and thin. The SEIs of the two treatment groups were much lower than those of the control group. There was no significant difference between the DMEM injection group and the untreated group.

　　 Masson trichrome staining method: Masson trichrome staining ADSCs and ADSCs-CM assembly. At 35 days postoperatively, the collagen fibers were arranged densely in the DMEM and untreated scar groups. In the ADSCs and ADSCs-CM groups, the deposition of fibers was good, and the collagen fibers were arranged well.

　　ADSCs are more effective than ADSCs-CM in reducing hypertrophic scars: ADSCs and ADSCs-CM can reduce scar hyperplasia. ADSCs is more effective than ADSCs-CM for further quantitative analysis of SEI value and mRNA level. The SEI value of ADSCs group was significantly lower than that of ADSCs-CM group. Compared with the ADSCs-CM group, the expression levels of α-SMA and type I collagen in the ADSCs group were significantly lower.

　　 ADSCs can be seen in the adipose stem cell injection treatment group: Before injection, ADSCs are labeled with Dil, a fluorescent fuel that tracks living cells. Frozen sections were examined with a fluorescence microscope. Three weeks after the initial treatment, a large number of green fluorescently labeled cells and blue fluorescent nuclei were evenly distributed in the adipose stem cells injected into the scar tissue. This may produce hypertrophic scar suppression.

　　 Conclusion: In this study, a rabbit ear hypertrophic scar model was used to experimentally study the anti-scarring effect of ADSCs and its conditioned medium on more than 140 wounds. It has been confirmed that ADSCs can inhibit the formation of hypertrophic scars by secreting a variety of anti-fibrotic cytokines when injected in vivo. In order to make it play a huge clinical application potential in the field of hypertrophic scar prevention and treatment, ADSCs as anti-scarring agents need further research.