| Objective:Human umbilical cord blood mesenchymal stem cells (HUCB-MSCs) without induction will be cultured in vitro, amplified, and then be transplanted into the streptozotocin-induced diabetic rats, observes the rats blood glucose, the amount of drinking water, body weight, the ratio of heart to body weight(HW/BW), the expression changes of the myocardial transforming growth factor beta1(TGF-β1)and the type-III collagen (collagenIII) , and myocardial ultrastructure changes to explore the protective effect of HUCB-MSCs on myocardium of diabetic rats.Methods:1. To culture and amplify HUCB-MSCs in vitro, by morphology and phenotype analysis and analysis to differentiate potential to identify cells. Till the sum of cell reaches 3×108, the MSCs from umbilical cord blood were marked with DAPI before implantation.2. Male Wistar rats were randomly divided into normal control group(NC),diabetes mellitus group(DM), diabetes with insulin glargine treatment group(DI),diabetes with stem cell treatment group(DS).The HUCB-MSCs marked by DAPI were transplanted into DS group by the caudal vein of rats, In DI group, insulin were injected through the abdominal for 6 weeks. Every group rats will be measured blood glucose, the amount of drinking water, body weight before making the model, before the treatment and after 6-week treatment. At 6th weekend, rats were killed, hearts were weighted and the HW/BW of all rats were calculated. Take the cardiac muscle organization to carry on the HE staining, Masson staining and PAS staining. Under the light microscope and the electron microscope observes the cardiac muscle pathology change and ultrastructure change. Immunohistochemical technique was used to evaluate the expression of TGF-β1 and the type-III collagen.Results:1. After the third generation of cultured and amplified HUCB-MSCs in vitro, HUCB-MSCs form homogeneous, long fusiform, showing the growth of fish-shaped. The DAPI marked dyes in the laser confocal scanning microscope to observe the cell nucleus coloration to send out the blue color fluorescence.2. Morphological observation: Through the HE, Masson and PAS staining of the myocardial tissue, showing DM group cardiac muscle arrangement disorder, obvious fiber to dissolve, the collagen fibers increased obviously, and had glycogen deposition. After treatment, DI group and DS group can be obvious prevention and reduce the cardiac muscle changes. The myocardial of DS group could be seen the blue fluorescent substances in frozen section.3. Ultrastructure: In DM group, myofibril arranged disorderly, the number of mitochondrial was increased and became swelling, while the changes of myocardial ultrastructure were obviously less worsened in DI group and DS group, especially in DI group.4. Expression of TGF-β1 and collagenIII: The expression of TGF-β1 and collagenIII in the two therapeutic groups were significantly lower than those in DM group (P<0.05). DI group and DS group were not significant difference (P>0.05).5.Blood Glucose: There were no significant differences among each group before making the model. After 1week of making the model, the blood glucose of DM group, DI group and DS group were significantly higher than that in NC group(P<0.05). After treatment, DI group's blood glucose compared with NC group's had no significant difference, while were significantly lower than that of DM group (P<0.05). DS group compared with DM group were significantly decreased, but DS group still higher than NC group and DI group (P<0.05). DI group, after the treatment, the blood glucose reduced obviously compared to before the treatment (P<0.05). DS group, after the treatment, the blood glucose also reduced obviously compared to before the treatment (P<0.05).6. Body Weight and HW/BW: There were no significant differences among each group before making the model. After that, NC group increased in body weight (P<0.05), while the remaining three groups were lower after 1week of making the model (P<0.05). After treatment, the weight of DI group and DS group were all markedly increased than that of DM group (P<0.05), especially in DI group. At the 6th weekend, HW/BW of DM group were significantly higher than that of NC group (P<0.05). HW/BW in all therapeutic group were lower than that of DM group (P<0.05), DI group and DS group were not significant difference (P>0.05).7. Amount of drinking water: There were no significant differences among each group before making the model. After 1week of making the model, the amount of drinking water of DM group, DI group and DS group were significantly higher than that in NC group(P<0.05). After treatment, the amount of DI group and DS group were all significantly decreased than that of DM group, while DI group were greatly lower than that of DS group, and higher than that of NC group (P<0.05). DI group, after the treatment, the amount of drinking water reduced obviously compared to before the treatment (P<0.05). DS group, after the treatment, the amount also reduced obviously compared to before the treatment (P<0.05).Conclusions:Human umbilical cord blood-derived mesenchymal stem cell can be transplanted into the injured cardiac muscle through the caudal vein, and they can degrade the levels of HW/BW, blood glucose and the amount of drinking water; depress the expression of TGF-β1 and collagen III; and they can alleviate the changes of morphological and ultrastructure in cardiac muscle, so they have the protective effect on myocardium of diabetic rats, but its effects worse than the treatment of insulin group. |
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