Isolation,Identification Of Mouse Umbilical Cord Mesenchymal Stem Cells And Transplantation For Mdx-Mice To Experimental Study On The Anti-inflammatory Effects

ObjectivesTo establish the isolation and the identification of mUCMSC (mouse umbilical cord stem cells, mUCMSCs); To study the functions about inflammatory of mUCMSCs treatments for X-linked muscular dystrophy (x chromosome-linked muscular dystrophy deficient, mdx) mice; To provide a proper theory fundament and technology for therapy of DMD in order to make this method into clinical earlier.Methods1. Isolation, culture and identification of mUCMSC:Collected umbilical cords from healthy pregnant C57BL/6(named C57 for abbreviation) mice when it is 16-17 days of pregnancy under the sterile environment. Cut the umbilical cords into small pieces, then adherent cultured in vitro and amplified to mUCMSCs. Identified the cells by their morphology, using flow cytometry, analyzing their characteristics of growth curve and their property to adipogenic, osteogenic and chondrogenic differentiation.2. Genotypic identification of DMD model mice:73 offspring mice from heterozygote mice were identificated by genotypic.50-100ul blood were collected from a mouse. DNA were extracted and genotypic identification was carry out by PCR-SSP (sequence specific primers polymerase chain reaction), and mdx mice were obtained for the experimental.3. MUCMSCs labeled with GFP and the distributions in vivo:the lentiviral containing GFP (green fluorescence protein) gene and mUCMSCs were cocultured,then mUCMSCs tagged by GFP intraperitoneal transplantation in mdx mice to observe mUCMSCs distributions in vivo by the small animal imaging instrument.4. Cellular transplantationtreatment experiment:mdx mice were divided into three groups randomly, respectively, each mouse intraperitoneal injected 0.4ml mUCMSCs saline suspension liquid (cell numbers were 5 ×104,5 ×105,5 ×106) treatment of mdx mice, once a week and four times in a row. At the same time set the negative control group and normal control group.5. Evaluation of anti-inflammatory treatment by mUCMSCs for mdx mice.(1) Observation the states of mdx mouse in 3 transplantation groups after treatment.(2) 1 week after received 4 times mUCMSCs treatment continuously, 100ul of blood in each mdx mouse was collected by cutting tail tip, when the separation of serum and blood cells can be seen, using automatic biochemical analyzer for the value of CK (creatine kinase) and comparative analysis of CK values between groups.(3) 1 week after received 4 times mUCMSCs treatment continuously,250-300ul of blood in each mdx mouse was collected by cutting tail tip, separated the serum out and using QAM-INF-1 protein chip to determination the inflammatory factors in peripheral blood of mdx mouse.(4) 1 week after received 4 times mUCMSCs treatment continuously, about lOug of gastrocnemius muscle weere collected, and after cleavage homogenizer to break and 4 ℃,14000rpm centrifugal for 10-15min. Supernatant 100ul of the liquid, inflammatory factors content were determined by QAM-INF-1 protein chip.(5) 1 week after received 4 times mUCMSCs treatment continuously, the gastrocnemius muscle tissue of mice was fixed with 4% formalin, and paraffin sections were made to observe the pathological changes of muscle tissue;(6) after the end of the experiment, the changes of the morphology and structure of the organs in the abdominal cavity were dissected and observed.6. Statistical methods:the experimental data using mean±standard deviation, using SPSS 17.0 statistical software analysis, used variance analysis, t test, correlation analysis and other methods to complete. P< 0.05 indicated that there was a significant difference, P< 0.01 indicated that there was a very significant difference.Results1. Isolation, culture and biological characters of mUCMSCs: Adhering culture the umbilical cord tissue of mice for about 24 hours, there were cells grown up from the edge of the tissue or scattered. Cell colony formation in 2-3 days, after passage cells grew faster than before and presented as the swirlings. Take the third generation mUCMSCs for flow cytometry and resulted out as highly expressed CD29, CD90, CD105 and low expression of CD34.The cells’growth curve showed a typical "s" type. The mUCMSCs had osteogenic and chondrogenic differentiation ability with in vitro lipid. In conclusion, we obtaining a good and homogeneous mUCMSCs.2.73 offspring mice were used for genotypic identification.23 mdx mice were used for this experiment.3.Transplanted the GFP labeled mUCMSCs into mdx mice and observed under imaging instrument.3h after transplantation, the fluorescent area was 35.2 mm2 initially and then 33.1mm2,24 hours after transplantation,the area of fluorescence was significantly reduced, the strength weaked,7 days after transplantation green fluorescence still could be observed in mouse peritoneal scattering distribution.4.Assessment of therapy effects after transplantation(1) Investigation of clinical manifestation after transplantation:The obvious clinical manifestation just like obviously dispiritedness, decreased activity, hair disorder, weight loss and other symptoms were not observed after mUCMSCs transplantation.(2) 1 week after received 4 times mUCMSCs treatment continuously, CK value of the groups which were injected 5×104,5×105,5×106 mUCMSCs by intraperitoneal were as follows,434.60±19.39 U/L,277.00±20.65 U/L,287.14±42.67 U/L, compared with the untreated group which CK value was 1374.60±127.13 U/L resulted as statistical significance (P< 0.05).(3)The results of protein chip technology showed that after once a week of mUCMSCs treatment continuous 4 times,14 factors had significant changes before and after treatment in 40 the candidate inflammatory cytokines in peripheral blood, including Etaxin, G-CSF, GM-CSF, IL-2, IL-4, INF-gamma, IL-5, IL-6, IL-17, MCP-1 MIP-1, PF4, TNFR-I gamma which were decreased after treatment, while ICAM-1 increased; 1 weeks after received 4 times of mUCMSCs treatment continuously, in mdx mice, IL-5, IL-6, IFN-γ, TNFR-1 were decreased with the cell dose increasing, the results showed that the cell treatment of mdx mice IFN-, IL-5, gamma IL-6, TNFR-1 in peripheral blood was negatively correlated with injection cell dose; 1 weeks after received 4 times of mUCMSCs treatment continuously, IL-5, IL-6, IFN-y, TNFR-1 in mdx mice was significantly lower than the control group mdx untreated mice; after the 4 times of mUCMSCs treatment CD30L, IL-21, MIP-la, PF4 in mdx were significantly lower than the control group mdx untreated mice.(4) Mdx gastrocnemius muscle by HE staining were observed under the microscope, infiltration of inflammatory cells were visible in mdx mouse muscle tissue. Muscle fiber sizes were different and partial dehiscence.The outline of those cells became round. Part of them were changed as heterogeneity. The nuclear in muscle cells increase or shifting into the center; gastrocnemius muscle fiber gaps were slightly widened. A small amount of fat, fiber connective tissue hyperplasia.1 week after receiving continuous four times mUCMSCs therapy, pathological changes in musle of mdx mice in three groups had improvement in different degrees. The inflammatory cells infiltration performanced decrease and the size of muscle cells tend to be consistent, and inflammatory cells infiltration decreased between muscle cells, cell size tends to unity. CNF(Centrally nucleated fiber) proportion in three treatment groups respectively for 89.37% ± 8.65,78.17% ± 6.65、73.81%±6.05 and compared with untreated group was 94.37%±11.65, the formor were less CNF and had statistical significance with the latter (P< 0.05).(5) The biological safety of cell transplantation,1 week after transplantation, the mdx mice in three groups of cell transplantation treatment were sacrificed by off neck and dissected. Watched the orgains in abdominal carefully and there were no abnormal tissue, so preliminary judgment that mUCMSCs treated mdx is safe.Conclusions1. MUCMSCs could be isolated from umbilical cord tissue of C57 mice by the whole mouse umbilical cord tissue adherent culture. MUCMSCs were highly expressed CD29, CD90, CD 105 and with a low expression of CD34. In vitro, mUCMSCs could differentiated into adipocytes, osteoblasts and chondrocytes.2. The mUCMSCs which were infected by lentivirus contained GFP genes transplanted to mdx mice after 7 days, which was proved that mUCMSCs could survive in the abdominal cavity of the mice.3. For 4 times intraperitoneal transplantation of 5×104-5×106 mUCMSCs for mdx mice, after that for 1 week, there were an improvement of the clinical symptoms of mdx mice; a significant reduction in CK value and alleviation of the pathological changes; significant reduction of the content of inflammatory factors in muscle tissue and serum; the expression of inflammation related factors reducing in the mdx mice peripheral blood, affecting IFN gamma, IL-5, IL-6, TNFR-1 and the hind limb muscle tissue pathological injury were alleviated partly, the mechanism of intraperitoneal injection mUCMSCs treated mdx mice may be related to relieving inflammation which the inflammatory cytokine mediated.