| Objective:To explore the effect of different glucose concentrations on the proliferation ofrat epidermal stem cells in vitro, and the impact of up-regulated β-catenin on theproliferation and differentiation of epidermal stem cells in diabetic rats. Observe β-catenin that up-regulated for the effects of diabetic rats wound healing. This studyprovides a new techology method for diabetes mellitus rats wound repairing,andestablishes furture experimental basis and theoretical basis for clinic application．Methods:(1) Mouse epidermal stem cells were separated and cultured by enzymedigestion combined with type IV collagen attachment method,and randomly dividedinto group A(control group,glucose concentration5.5mmol/L), group B(glucoseconcentration10mmol/L),group C(glucose concentration20mmol/L),and groupD(glucose concentration40mmol/L). The morphology of epidermal stem cells wereobserved by microscope;the expression of β-catenin and Wnt1were tested byimmunocy-tochemistry; The average integral absorbance values of positive cells weremeasured with the image analysis software.(2) Diabetes mellitus rat model was made by intraperitoneal injection ofstreptozocin. ESCs of diabetic SD rats were isolated and cultured in vitro,and dividedinto transfection group(pcDNA3.1myc-β-catenin plasmid was transfected into ESCsthrough liposome-mediating method), liposome group (liposome was transfected intoESCs as control)and control group(normal culture). Expression of β-catenin in threegroups were tested by immunocytochemistry; the expression levels of β-catenin、Wnt1、CyclinD1in transfection group and control group were detected by Westerrnblotting.(3) Taking15SD rats successful models,causing the diameter of2.1cmfull-thickness dermal wounds by a special punch in the back on both sides of thespine. Four wounds of each rat were randomly divided into four groups: group A(Human amniotic membrane(HAM) loading ESCs of diabetic rats which β-catenin was up-regulated were implanted), group B(HAM loading ESCs of diabetic rats wereimplanted),group C(HAM was implanted to wound)and group D(blank control group,without any intervention). General situation of wound healing was observed,and thehealing rate of wound was calculated. Hemat-oxylin and eosin staining andimmunohistochemical staining of (SP method) were used to detect expression ofproliferating cell nuclear antigen in healing tissue.Results:(1) With the glucose concentration increased, the number of cells significantlydecreased,cell growth slowed,and cell vitality reduced; Immunocytochemistryanalysis showed that, the expression of β-catenin and Wnt1in group C(0.079±0.007;0.083±0.004)and group D(0.068±0.008;0.070±0.006) were significantlylower than the control group (0.096±0.123;0.095±0.010), p <0.01.(2) Immunocytochemistry results show that the expression level of β-catenin intransfection group was higher than the liposome group and control group (p <0.01);expression levels of β-catenin, Wnt1and CyclinD1in transfection group and controlgroup were respectively(1.451±0.027,1.094±0.026),(1.084±0.078,0.694±0.082),(1.490±0.030,0.807±0.019),and the difference between transfection group and controlgroup was statistically significant(p<0.01).(3) Compared with group B, C, D, the wound healing rate of group A wassignificantly higher (P <0.01). PCNA-positive cells could be seen in wound tissue ofeach group, but the expression of PCNA-positive cells in group A was higher than theother three groups.Conclusion:(1) Under the condition of this experiment, high glucose can inhibit the grouth ofepidermal stem cells in vitro, and the expression of β-catenin and Wnt1in epidermalstem cells decreased.(2) pcDNA3.1myc-β-catenin plasmid can successfully transfected into ESCs ofdiabetic SD rats through liposome-mediating method; the expression of β-cateninincreased in transfected cells, accompanied by the expression of Wnt1and CyclinD1increased.(3) Epidermal stem cells which β-catenin was up-regulated in diabetic rats may contribute to the healing of diabetic impaired wound．... |
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