The Promotive Role Of UCMSCs And ECFCs Transplantation And Culture Matrix On Wound Healing In Diabetic Mice

Objective:The aims of our study are to confirm and to compare the effectiveness of the treatment of diabetes mice skin trauma with umbilical cord mesenchymal stem cells(UCMSCs), endothelial colony-forming cells(ECFCs), their culture matrix and the combination of UCMSCs and ECFCs. Observe the promotive effect of treatment of diabetic wound with a single cell, culture matrix and combined the two cells and discuss the effect of related mechanisms.Methods:1. To establish a diabetic mice skin trauma model:Feeding36mice which were seven weeks old on the appropriate environment. Weighing and measuring blood glucose(>16.7mmol/L) after a week. Making circular wound on back in mice with hole puncher which diameter is6mm after anesthetization.2. Mice were divided randomly into six groups and there were six mice in each group:group A:ECFCs,group B: ECFC-CM (conditioned matrix), group C:MSCs which from human umbilical cord,group D:MSC-CM (conditioned matrix),group E:the mixed cell of ECFCs and UCMSCs, group F:the normal group which use to compare with other groups(PBS).3. Cell transplantation:After the success of establishing model, using multipoint subcutaneous injection method to inject ECFCs and UCMSCs into the place which next to the wound three millimeter for four sites on group A and group B.(total cells injection1*10^6per mice, total:100ul). Inject the culture supernatant of ECFCs and UCMSCs of the same number of cells cultivated by M199into the identical place on group C and group D using the same method. Inject ECFCs and UCMSCs into subcutaneous tissue (0.5*10^6ECFCs mixed with0.5*10^6UCMSCs) on group E.Inject PBS on group F(100ul per mice). After transplantation,use transparent hydrocolloid dressing to cover the wound.4. Get materials and observe:Take pictures of all the surface of the wound in mice use camera and record the process of wound healing, the blood glucose and body weight at the same time.After18days of the transplantation of cells and nutrient medium,get material from the skin tissue of the mice on group ECFCs and UCMSCs and use them to make frozen section.Afterwards, observe the specific location of the transplantation cells which were marked with the CM-Dil under the fluorescence microscope; observe the density of new capillaries of the skin tissue with H-E staining and vWF immunohistochemical method; using Western Blot method to detect angiogenesis factors and the expression of signal pathway protein.Results:After transplantation of cells and culture matrix, monitoring the weight and blood glucose of mice randomly every2days. As a result, the weight of mice in groups increased gradually with the extension of time, and has no obvious difference between groups (P>0.05); Blood glucose were greater than16.7mmol/L, and has no obvious difference between groups (P>0.05).Bright red,granular,soft and wet granulation tissue had been formed gradually in the wound edge, which filled the wound tissue defect.Transplanted cells group and CM group significantly shorten time healing the wound compared with control group (PBS), the fastest group in wound healing is the mixed cell group,the healing rate of which have statistical difference compared to other groups(P<0.05), ECFC、ECFC-CM, UCMSCs, and MSC-CM had no obvious difference (P>0.05) in healing rate, while they have shorter time than the PBS control group. After cells and autocrine factors had been transplanted for18days,detecting proteins expression of VEGF, AKT, pAKT, ERK1/2in the wounded skin tissue using Western Blot, the results showed:the expression of VEGF in ECFCs, ECFC-CM, UCMSCs, UCMSC-CM and combined transplantation group was obviously higher than that of PBSs group, the difference was statistical significance(single transplantation group (P<0.05), combined transplantation group (.P<0.01)), the cells and autocrine transplantation could increase the secretion of VEGF. The VEGF expression of the combined transplantation group is higher than single transplantation group, the difference was statistically significant (P<0.01), indicating the highest amount of VEGF expression after the joint cell transplantation. The VEGF expression in autocrine factors group and single cell transplantation group had no obvious changes, and there was no difference between groups (P>0.05). And the expression of signal pathway proteins AKT, pAKT, ERK1/2suggested that whose expression of ECFCs, ECFC-CM, UCMSCs, UCMSC-CM and combined transplantation group was obviously higher than PBSs group, the difference was statistically significant (compared between the single transplantation group and the PBS group, P<0.05; compared between the transplantation group and the PBS group, P<0.01); The combined transplantation group had the highest protein expression,compared with single transplantation group, the difference was statistically significant (P<0.01); Compared between single transplantation groups turned out to be no difference (P>0.05).These indicate that autocrine VEGF maybe play an important role in promotion of angiogenesis and tissue repair by ECFCs and UCMSCs. Combined cell transplantation may be more conductive to the VEGF secretion and promote angiogenesis and tissue repair.Conclision:1. The transplantation of UCMSCs, ECFCs and its autocrine factors can promote the formation of new blood vessels effectively and accelerate tissue repair.2. The combination transplantation of UCMSCs and ECFCs has synergy to promote the formation of new blood and accelerate tissue repair more significantly than transplanting only one of the cells.3. The promotive effect of the ECFCs and its autocrine factors on wound healing in diabetic mice can be achieved the same effective role compared with the UCMSCs and its autocrine factors, provides a new thought for the treatment of tissue repair by cell transplantation in clinical.