| Objectives The purpose of this study is to investigate the effect of mi R-21 on promoting UCBMSCs to differentiate into vascular and the vascular regeneration function of mi R-21 in the hind limb ischemia of nude mouse model.Methods 1: UCBMSCs(suppoted by Tongji University stem cell bank) was cultivated and induced to form bone, fat and cartilage to identify the multiple differentiation characteristic. 2: Building up Lenti-Lacz-Luciferase and Lenti-mi R-21-Luciferase Lentiviral vectors, determining its MOI value. At the 4th day after Lentiviral transferred to UCBMSCs, the cell were treated with D-Luciferin Potassium salt, then BLI was used to detect Luciferase and detect Luciferase and determine the transfer efficiency. 3: At 0,1,4,7,14 and 28 th after target gene transferred to UCBMSCs, the Real time PCR and Western Blot were used to detect the key vascular forming factors and target genes. 4: After Lenti-Lacz-Luciferase and Lenti-mi R-21-Luciferase transferred to P3 UCBMSCs,the cells were inoculated in a96 hole plate which contains Matrigel. During 1 to 4th, the tubes were observed by microscope and the length of the tubes were took for quantitative analysis by Olympus Cell R imaging. 5: To build up a CLI model, we ligated the femoral vascular and popliteal vascular of nude. The gluteus maximus and gastrocnemius were injected with 100μL UCBMSCs/mi R-21, UCBMSCs / Lacz and Saline respectively. Thermal imaging technology was used to trace the transplanted cells `s positions, and Laser Doppler ultrasonography was used to determine vascular regeneration. The specimen were harvested in 28 th. Lower limb vascular was perfused by macrofil. Gluteus maximus and gastrocnemius were treated with 4% paraformaldehyde, then then dehydrated, paraffin-embedded and made in slices. HE and CD31 immunofluorescence staining were used to detect the new blood vessels.Results 1: At 28 th after Osteogenic induction, calcium nodules can be detected in ARS staining. At 16 th after cartilage induction and fat induction, cells showed positive expression in Alcian Blue and Oil Red O staining. These results prove that UCBMSCs have characteristics of stem cell. 2: Lentiviral vector was successfully constructed. 3:The results of q PCR show that HIF-1α and VEGF in mi R-21 group express strongly at 4th, and this phenomenon continues until 14 th. Other key vascular forming factors such as SDF-1,b FGF,PLGF and SCF show the same tendency as HIF-1α and VEGF.In addition, the results of Western Blot and q PCR are similar. 4: Results of Matrigel show that Lenti-mi R-21- Luciferase group has the most tube structures and the longest tube length in three parallel experiment groups. 5: Results of BIL show that stem cell which transferred to CLI zone stay in place. At 3th,the fluorescence reach the highest expression, sustain expression until 28 th. The results of Laser Doppler show that vascular perfusion in the experimental group was significantly better than the control group in the first three days. This result keeps the same until 28 th.Results of Immunofluorescence show that amount of CD31 expression in mi R-21 group is higher than Lacz group(3 times) and saline group( 20 times). The result keeps up with early Doppler observation.Conclusions mi R-21 can promote UCBMSCs differentiate into vascular and have a therapeutic effect in CLI. |
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