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Objective@#To investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats.@*Methods@#(1) Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site (HLA-DR). The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. (2) The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. (3) Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 μg/mL exosomes group, 50 μg/mL exosomes group, and 100 μg/mL exosomes group according to the random number table (with 6 wounds in each group), and a total volume of 100 μL phosphate buffered saline, 25 μg/mL exosomes, 50 μg/mL exosomes, and 100 μg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day (PID) 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson staining. Data were processed with analysis of variance for repeated measurement, analysis of variance of randomized block design, one-way analysis of variance, and Bonferroni test.@*Results@#(1) The cells, which were isolated and cultured, displayed typical cobblestone morphology with many microvilli on cell surface. Among the cells, the positive expression rates of CD29, CD90, SSEA3, and SSEA4 were above 50.0%, and the rate of CD105 was 8.0%, while the rates of CD31, CD34, and HLA-DR were almost 0. The cells could differentiate into adipocytes and osteoblasts. The above results revealed that the cells cultured were human amniotic epithelial stem cells. (2) Human amniotic epithelial stem cells-derived exosomes were round or oval vesicles with diameter from 50 to 150 nm. (3) On PID 7 and 21, wound healing rates of the four groups were close (with P values above 0.05). On PID 14, wound healing rates of 50 and 100 μg/mL exosomes groups were (89.8±4.3)% and (92.0±4.6)% respectively, significantly higher than the wound healing rate of control group [(80.3±6.4)%, P<0.05 or P<0.01]. Moreover, the wound healing rate of 100 μg/mL exosomes group was significantly higher than that of 25 μg/mL exosomes group [(83.3±5.1)%, P<0.05]. On PID 21, the numbers of skin accessories in 50 and 100 μg/mL exosomes groups were 4.3±1.4 and 5.1±1.6 respectively, obviously more than those of control group and 25 μg/mL exosomes group (respectively 1.4±0.5 and 1.8±0.6, with P values below 0.01). Well reorganized collagen fibers were observed just in the healed wound tissue of 50 and 100 μg/mL exosomes groups.@*Conclusions@#Human amniotic epithelial stem cells-derived exosomes can promote healing of wound with full-thickness skin defect in rats.
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BACKGROUND:Gastric cancer mesenchyal stem cel s from clinical stomach cancer specimens and tumorigenic tissues in nude mice are similar to the bone marrow mesenchymal stem cel s in biological characteristics, which have been proved to be an important component of tumor microenvironment to promote tumor growth. It is speculated that biological characteristic of bone marrow mesenchymal stem cel s may change in stomach cancer microenvironment. OBJECTIVE:To observe the effect of stomach cancer microenvironment on morphology and proliferation of bone marrow mesenchymal stem cel s and expressions of CD34 and CD44. METHODS:Rat bone marrow mesenchymal stem cel s were cultured alone as control group. In the test group, rat bone marrow mesenchymal stem cel s were co-cultured with human stomach cancer BGC-823 cel s using Transwel chamber assay to establish the stomach cancer microenvironment. Then, cel morphology, proliferation, cel cycle and CD34, CD44 expressions were observed and detected using inverted phase contrast microscope, MTT assay, and flow cytometry, respectively. RESULTS AND CONCLUSION:In the test group, bone marrow mesenchymal stem cel s were similar to human stomach cancer cel s BGC-823 that arranged disorderly and irregularly, were interconnected loosely, became thinner and longer, and grew in clusters with smal er nuclei. The cel proportion in G 1 phase significantly decreased, but that in S and G 2/M phases significantly increased (P<0.01, P<0.05). The positive rate of CD44 significantly declined, and the CD34 expression significantly raised (P<0.01). In conclusion, stomach cancer microenvironment by non-contact co-culture with BCG-823 cel s has an obvious effect on the morphology, proliferation and surface antigens expressions of bone marrow mesenchymal stem cel s that wil tend to be malignant gastric cancer cel s.