| Objective:The normal adipose derived stem cells(n ASCs)and diabetic Adipose-derived stem cells(d ASCs)were isolated and cultured.The morphology,surface markers,proliferation and differentiation of the two kinds of cells were comparatively studied in vitro.The wound healing of pressure ulcers after two kinds of cell transplantation was compared in vitro.To investigate whether d ASCs retain the ability of promoting wound healing and provide experimental evidence for autologous transplantation of d ASCs in the treatment of chronic wounds.Methods:1.Adipose tissue are derived from the patients from Affiliated Hospital of Zunyi Medical College of Burn and Plastic Surgery.After informed consent,adipose tissue from diabetic and non-diabetic patients is obtained during surgery under sterile conditions.Adipose-derived stem cells(ASCs)were extracted by collagenase digestion,cultured in low glucose DMEM medium containing fetal bovine serum and passaged by trypsin digestion.The morphological changes of the cells from two different sources were observed under an inverted microscope,and the expression of the surface markers of the third generation cells was detected by flow cytometry.2.The third generation of n ASCs and d ASCs were taken as experimental cells,and the proliferation ability of the two kinds of cells was measured by MTT assay and the growth curve was drawn.The two kinds of cells were induced to adipogenesis and osteogenesis respectively.After 21 days,the adipogenic and osteogenic conditions were observed by oil red and alizarin red staining,respectively.Real-time fluorescent quantitative PCR was used to detect the expression of PPAR-? and BMP-2 and BGP m RNA in adipocytes and osteoblasts two weeks after.3.Animal models of skin pressure ulcers in mice back were established and randomly divided into three groups: n ASCs transplantation group,d ASCs transplantation group and PBS group(Normal control group,NC),8 mice in each group.The third generation of cells in the proliferative phase were transfused with virus-coated green fluorescent protein(GFP)and then injected into the wounds of each pressure ulcer at 1 × 10 6 cells.The PBS group was injected with the same volume of PBS.Then observe the pressure ulcer wound healing.The mice were sacrificed 11 days and 21 days after injection.The wounds of the pressure ulcer were dissected and stained with HE and Masson.The thickness of epidermis,infiltration of inflammatory cells in wound tissues and collagen deposition were observed.CD31 and S100 immunohistochemical staining were used to observe the condition of blood vessel regeneration and nerve regeneration.At day 11,The two kinds of cell colonization and survival were tracked.Results:1?ASCs were successfully isolated from adipose tissue of normal and diabetic patients.The adherent cells were observed 8 hours after primary inoculation,and the growth of cells was accelerated after passage.Both of the third generation cells were uniform in shape,mostly fusiform in shape,Growth,no significant difference in morphology.Flow cytometry detected two kinds of third generation cells CD90,CD105,CD73,CD44 were positive expression,CD34 was negative expression.2.The results of MTT assay showed that the proliferation ability of n ASCs was stronger than that of d ASCs and the growth rate was fast.The induced differentiation showed that both n ASCs and d ASCs could be induced to adipogenesis and osteogenesis.The adipogenic capacity of d ASCs was stronger than that of n ASCs,but the osteogenic capacity of d ASCs was lower than that of n ASCs.Real-time quantitative PCR results showed that the expression of adipogenesis gene PPAR-?m RNA in d ASCs was higher than that in n ASCs after induction,whereas osteogenesis gene BMP-2 and BGP m RNA expression was lower than that in n ASCs.The differences were statistically significant(P <0.001).3.Successfully established the mouse back skin pressure ulcer model.The wound healing rate of n ASCs group was 36.47 ± 5.83%,89.18 ± 2.14% and 99.47 ± 0.89% on the 5th,9th and 13 th day after the cell transplantation respectively,while the d ASCs group was 35.66 ±6.52%,60.40 ± 7.67% and 87.18 ± 2.56,PBS group was 19.89 ± 7.20%,46.32 ± 4.21% and 66.25 ± 5.26% respectively,which indicated that the wound healing of nASCs group was the fastest,the healing rate of d ASCs group was the second,the PBS group was slowest.On the eleventh day,HE staining showed that the n ASCs group began to have epithelialization,while the d ASCs group and the PBS group were separated from the epidermis and basal tissue.The degree of inflammatory cell infiltration in the n ASCs group,the d ASCs group,and the PBS group decreased in turn.The difference was statistically significant(P<0.001).On the 21 st day,the degree of inflammatory cell infiltration was not significantly different among the three groups;the epidermal thickness was gradually decreased in the n ASCs group,the d ASCs group,and the PBS group,and the difference between the groups was statistically significant(P<0.05).In the three groups,the regenerated hair follicles were seen in the periphery of the wound,in which the nascent follicles were seen in the center of d ASCs group.Masson staining showed that collagen deposition in d ASCs group(48.38 ± 1.36%)was more than that in n ASCs group(45.85 ±1.95%)on day 11,but there was no statistical significance.Collagen deposition in d ASCs group and n ASCs group was significantly greater than that in PBS group(41.46 ± 1.75%),with statistical significance(P <0.001).On the 21 th day,the collagen deposition in n ASCs group(56.93 ± 1.67%),d ASCs group(54.17 ± 1.71%)and PBS group(50.34 ± 1.29%)decreased gradually,the difference between groups was statistically significant(P<0.05).The results of CD31 immunohistochemistry showed that there was no significant difference between the n ASCs group(17.80 ± 0.84)and the d ASCs group(17.20 ± 1.30)on the 11 th day.The number of blood vessels in n ASCs group and d ASCs group was significantly higher than that in PBS group(8.00 ± 1.41),the difference was statistically significant(P <0.001).On the 21 th day,the number of blood vessels decreased in the n ASCs group(20.00 ± 1.87),d ASCs group(18.00 ± 2.12)and PBS group(14.00 ± 1.58),the differences among the groups were statistically significant(P <0.05).The results of S100 immunohistochemistry showed that on the 11 th day,no positive chromogenic cells were observed in the n ASCs and d ASCs groups.On the 21 th day,the Schwann cell density in the n ASCs group(1.63±0.08)was lower than that in the d ASCs group(1.76±0.12).However,it was not statistically significant.the n ASCs group and d ASCs group were significantly larger than the PBS group(0.55±0.04),and the difference was statistically significant(P<0.001).Wound cell tracing results showed that both n ASCs and d ASCs could be colonized in the wounded skin after 11 days.Conclusion:1.Compared with n ASCs,the morphology and surface markers of d ASCs showed no significant changes,their proliferation and osteogenic differentiation ability decreased,while adipogenic differentiation ability increased.2.Local transplantation of d ASCs can promote wound healing by regulating local inflammation,increasing collagen deposition on the wound,and promoting angiogenesis and nerve regeneration.3.The ability of d ASCs to promote wound healing was lower than n ASCs. |
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