Therapy And Comparison The Treatment Of Human Cord Blood Mononuclear Cells And Umbilical Cord Mesenchymal Stem Cells On Rat Denervated Gastrocnemius Muscles Damage

Obj ective:1.To explore the therapeutic effects of human cord blood mononuclear cells(CB-MNCs)and umbilical cord mesenchymal stem cells(UC-MSCs)on rat gastrocnemius denervation.2.To compare the therapeutic effects of CB-MNCs and UC-MSCs on the denervated nerve injury of rat gastrocnemius,and provide the experimental evidence for the best clinical treatment.Methods:1.Preparation of human cord blood mononuclear cells(CB-MNCs)and umbilical cord mesenchymal stem cells(UC-MSCs)1.1 Isolation,identification and preparation of CB-MNCs:Fresh umbilical cord blood with the lymphocyte separation solution in 1:1 ratio was gradiently centrifuged to obtain mononuclear cells.The cells was washed by physiological saline three times.Cell viability was detected by trypan blue exclusion test;cell phenotype was detected by flow cytometry.The density of cells was adjusted to 1×106 in 1mL saline for injection and applied within 1 hours,while the remaining cells were frozen in liquid nitrogen for later use.1.2 Isolation,culture and identification of UC-MSCs:Fresh umbilical cord was cut into small pieces and adhered to culture mesenchymal stem cells.When cultured to the third generation,the morphology of the cells was observed under microscope.The cell phenotypes were detected by flow cytometry.The cell differentiation identification by alizarin red staining,oil red O staining and alexin blue staining were carried out after cells respectively differentiated in osteogenic,adipogenic and chondrogenic induced mediums.The cells were cultured until the fifth passage and cryopreserved.2.The model of rat gastrocnemius denervation injury:Forty juvenile Wistar rats were randomly divided into normal group(Normal group,n=10)and sciatic nerve disconnection model groups(Model group,n=30).The sciatic nerves on both sides of the Model group were cut to create a 1 cm length nerve defect.The Model group further was divided into CB-MNCs treatment group(MNC group),UC-MSCs treatment group(MSC group)and saline treatment group(NS group),with 10 rats in each group.3.Cell therapy:On d7,d14 and d21 after sciatic nerve injury,the different groups were treated as follows:?In the MNC group,106 CB-MNCs were diluted in 1 mL of normal saline,and each side of the muscle abdomen received 0.5 mL injection which divided into 3 injection points.?In the MSC group,106 UC-MSCs were diluted in the same way and injected into the gastrocnemius muscles.?The muscles of rats in the NS group received an equal volume of saline injection.The general conditions of rats were observed in the transplant intermission.On d28 of modeling,the body of rats was weighed.The blood in heart was taken for examination after anaesthesia,and both gastrocnemius muscles were taken out.The wet weight of muscles was weighed and the ratio of gastrocnemius/body mass was calculated.Some part of muscle tissues was fixed with fixative solution,and the remaining tissues were stored at-80 ?for mRNA and protein detection.4.Efficacy assessment4.1 The rat muscle wet weight ratio in each group was analyzed statistically.The area of gangrene in both sides of the arch was measured and the proportion of gangrene in the arch was calculated.The fixed gastrocnemius muscle was embedded in paraffin and sectioned.HE staining was used to measure fiber cross-sectional areas in each group.4.2 Serum was taken from the heart blood after centrifugation.Appropriate amount of muscle tissues were taken to make tissue homogenization.The biochemical test kits were used to detect the content of creatine kinase(CK),malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT)in serum and tissue homogenization of each group.4.3 RNA from muscles in each group was extracted,and the expressions of apoptosis-related protein genes(Bcl-2,Bax,Caspase-3),inflammatory factor protein genes(TNF-?,IL-10)and muscle tissue-associated protein genes(VEGF-?,?-actin,Dystrophin)were measured by real time quantitative polymerase chain reaction(RT-qPCR).4.4 The muscle tissue proteins of each group were extracted,and the expressions of VEGF-?,?-actin and Dystrophin were detected by Western blot.4.5 Paraffin sections of muscle tissues were immunohistochemically stained with Dystrophin to further verify the difference in the groups.Results:1.Acquisition,culture and identification of CB-MNCs and UC-MSCs1.1 The umbilical cord blood was centrifuged to obtain CB-MNCs.The survival rate of CB-MNCs using the trypan blue staining was greater than 95%.The cell survival rate after resuscitation was about 60-70%.The positive expression rate of CD34 in the cell group was 1.4-1.9%by flow cytometry.1.2 Long spindle-shaped adherent cells were successfully isolated and cultured which grew into a vortex from the umbilical cord.They highly and positively expressed CD105,CD90,CD73,CD29 and CD44 with low and nagetive expression of CD31,CD34 and CD45 by flow cytometry.After being cultured in differentiation mediums,the cells could differentiate into osteogenesis,adipogenic and cartilage.The above characteristics were in accordance with the definition of MSCs,proving that the cultured cells were MSCs.2.Animal behaviors during modeling period:Compared with the Normal group,the rats in Model group showed that the hind limbs were paralyzed and towed with drooping toes after the sciatic nerves were cut.Two weeks later,the feet edema were obvious,and the gangrene of the arches and broken toes began to appear.There was no animal death.The above results indicated a successful model with a 100%success rate.3.Therapy of rat denetvated gastrocnemius muscles damage using CB-MNCs and UC-MSCs and differences in efficacy between the two cells3.1 On d28,the area ratio of the arch gangrene in Model group was measured.The ratios in cell treatment groups were smaller than that in the NS group(P<0.05),whilt the area of the gangrene between the cell treatment groups was no significant difference(P>0.05).The muscle atrophy in NS group was the most obvious compared with Normal group and cell treatment groups.The muscles were thin with a dim color in NS group.Comparing the wet weight ratio of the gastrocnemius muscles in each group,model groups were smaller than that in Normal group(P<0.05),but the ratios of the cell treatment groups were higher than that of NS group(P<0.05),and MSC group was higher than MNC group(P<0.05)thereinto.In comparison with the other groups,the NS group had the most thinnest muscle fibers,and the connective tissue area between the muscle bundles became larger.The cross-sectional areas of gastrocnemius muscles in Model group were smaller than that in Normal group(P<0.05).Among the model groups the cross-sectional area of NS group was the smallest(P<0.05),and MSC group was larger than MNC group(P<0.05).3.2 The CK and MDA contents in the serum and muscles of the cell treatment groups were significantly lower than those in NS group(P<0.05),and the MSC group was much lower than the MNC group(P<0.05);the contents of CAT and SOD in cell treatment groups increased significantly(P<0.05),and MSC group was higher than MNC group(P<0.05)detected by biochemical kits.3.3 Real-time quantitative PCR showed that the mRNA expressions of Caspase-3,and Bax were lower in cell treatment groups than in NS group(P<0.05),and MSC group was significantly lower than MNC group(P<0.05).The mRNA expression levels of Bcl-2,a-actin and VEGF-a in cell treatment groups were increased compared with NS group(P<0.05),and the expression of the above genes in MSC group were higher than those in MNC group(P<0.05).The mRNA levels of TNF-a and Dystrophin in Model group were higher than those in the Normal group(P<0.05),and the expressions in the MNC group and the MSC group were higher than those in the NS group(P<0.05).While between the two cell treatment group,the differences were not statistically significant(P>0.05).The mRNA expression of IL-10 was increased in the Model group,and the cell treatment groups were higher than the NS group(P<0.05),whilt the difference was not statistically significant between the cell treatment groups(P>0.05).3.4 Western blot analysis showed that the expressions of a-actin and VEGF-a proteins in the cell treatment groups increased highly than in NS group(P<0.05),and MSC group was higher than MNC group(P<0.05).The content of Dystrophin in the Model group was higher than that in the Normal group,and the cell treatment groups were higher than the NS group(P<0.05),whilt the difference was not statistically significant between the MNC and MSC groups(P>0.05).3.5 Immunohistochemistry showed that the expression of Dystrophin in model groups was higher than that in Normal group(P<0.05).But among the Model group,the cell treatment groups were significantly higher compared with the NS group(P<0.05).There was no difference in protein expression between the two cell treatment groups(P>0.05)Conclusions:1.Injecting CB-MNCs and UC-MSCs into the paralyzed gastrocnemius can effectively reduce the proportion of apoptotic cells,promote the wound healing,relieve muscle atrophy,and improve muscle functions.2.The therapeutic effects of UC-MSCs on the denervated nerve injury of the gastrocnemius are better than those of CB-MNCs,and it can be used as an auxiliary treatment before clinical repair of nerve injury.