| Chapter One Preparation and Detection of Mesenchymal Stem Cells and Diabetic Foot Mouse ModelObjective:To prepare highly purified umbilical cord mesenchymal stem cells by collecting and culturing umbilical cord.A diabetic foot mouse model was constructed by unilateral limb femoral artery and its branches ligation in db/db mice.The prepared mesenchymal stem cells and diabetic foot model mice were also examined.Methods:Human umbilical cord mesenchymal stem cells were cultured by tissue explant.Detected by 24-hour cell viability,cell surface markers,etc.Diabetic foot mouse model was established by ligating unilateral limb femoral artery and its branches in db/db mice,and the model was detected by clinical manifestations,random blood glucose,and limb temperature.Results:Human umbilical cord mesenchymal stem cells were successfully cultured by tissue block method.The cell survival rate at 24 hours was 91.2±0.86%,which was greater than 90%.Cell surface markers:There was a high proportion of CD73(91.63%),CD90(99.71%),and CD105(99.94%)positives,all of which were more than 90%,while CD34 expression was low(0.64%).Cell viability and cell surface markers met the requirements for stem cells.By ligature Thirty diabetic foot model mice were successfully constructed by free unilateral femoral artery and its branches in db/db diabetic mice.The random blood glucose of PBS MSC group was 32.72±0.70 mmol/L and 32.78 ± 0.98 mmol/L,respectively,and the ratio of limb to limb was 32.84±2.28%and 31.44±1.21%,respectively.The requirements for modeling the diabetic foot model were met.Conclusions:Human umbilical cord mesenchymal stem cells can be successfully prepared by tissue explant.Diabetic foot model mice can be successfully constructed by ligating the femoral artery and its branches in db/db diabetic mice.Chapter Two Mouse Model of Diabetic Foot Treated with Umbilical Cord Mesenchymal Stem CellsObjective:To evaluate the therapeutic effect of cultured umbilical cord mesenchymal stem cells transplanted into diabetic foot model mice.Methods:Thirty diabetic foot model mice were randomly divided into PBS group(15 mice)and MSC group(15 mice).1 × 10 intramuscularly transplanted stem cells at the MSC site 6 1 mL total.The PBS group was injected with the same volume of PBS solution in the same way.Body temperature changes,blood glucose changes,and body weight changes were measured before and 1 week,2 weeks,and 3 weeks after the intervention.Capillary density was observed by immunofluorescence staining.HE staining was used to observe the changes of muscle tissue.Western blotting was used to detect the changes of Bax,Bcl-2 and VEGF after cell transplantation.Results:After intervention,the blood glucose of MSC group was significantly decreased compared with PBS group(P<0.05),and the body weight of both groups was decreased after intervention(P>0.05).Limb temperature increased significantly in the MSC group compared with the PBS group after intervention(P<0.05).The results of immunofluorescence staining showed that the vascular density of MSC was higher than that of PBS group at 7 days,14 days and 21 days after intervention(P<0.05).The results of HE staining showed that the tissue of PBS group was significantly atrophied after intervention,and a large number of cells were degenerated or even apoptotic to varying degrees.However,in the MSC group,the muscle tissue morphology was regular and intact,and more new capillary formation was observed.The results of Bax,Bcl-2 and VEGF by Western blot showed a statistically significant difference in VEGF,Bcl-2,and Bax in the MSC group(P<0.05).Conclusions:Transplantation of stem cells into diabetic foot model mice can significantly improve limb temperature,increase capillary density and promote muscle tissue regeneration.The increase of VEGF and Bcl-2 and the decrease of Bax suggest that stem cells may play an important role in inhibiting apoptosis and promoting cell regeneration. |
PDF Full Text Request