| With the increasing number of ischemic diseases,various ischemic diseases have become one of the diseases with the highest disability and mortality rates in the world,and research on stem cell therapy may bring new hope to patients.Endothelial progenitor cells(EPCs)-based stem cell transplantation can effectively protect the integrity and function of ischemic tissue.Angiogenesis refers to the formation of new blood vessels in a budding or non-budding manner on the basis of the original capillaries and(or)microvenous veins,which involves the proliferation and migration of mature vascular endothelial cells;endothelial cell proliferation and migration play an important role in angiogenesis.Signal transduction and transcription activation factor 3(STAT3)promotes the proliferation and differentiation of EPCs into endothelial cells through vascular endothelial growth factor(VEGF)on the one hand,and promotes the activation of Wnt-5a / ?-catenin pathway on the other hand,It is beneficial to vascular endothelial cell-mediated angiogenesis,and then relieves lower limb ischemia.Studies have confirmed that mi R-320 is a class of mi RNAs with significant anti-angiogenic effects.Taurine up-regulated gene 1(TUG1)is an lnc RNA that is conserved in vascular endothelial cells and plays an important role in promoting proliferation and migration of vascular endothelial cells and inhibiting their apoptosis,thereby promoting angiogenesis.First of all this experiment studied the expression changes of lncRNA TUG1,mi R-320,STAT3,VEGF,Wnt-5a,and ?-catenin in different ischemic time in animals;then cultured EPCs to study their proliferation,migration and angiogenic ability.Finally,it was transplanted into lower limb ischemic nude mice to confirm its improvement on ischemia,and the relationship between the improvement of ischemia and the expression of lnc RNA TUG1,mi R-320,STAT3,VEGF,Wnt-5a,?-catenin was compared.Part 1 Tissue damage of lower limb ischemia at different timeObjective:To investigate the expression of lncRNA TUG1,mi R-320,STAT3,VEGF,Wnt-5a and ?-catenin in nude mouse models of lower limb ischemia and the changes of ischemic time.Methods:Twenty 6-week-old nude mice were randomly divided into 5 groups,4 in each group:(1)48h ischemia group;(2)24h ischemia;(3)12h ischemia;(4)6h ischemia group;Each group was fed with feed for 4 weeks,and the weight of nude mice was monitored to establish a model of lower limb ischemia in nude mice.After 48 hours,the changes of blood flow in the ischemic lower limbs were detected by laser Doppler,then the lower limbs were dissected,the gastrocnemius muscle was isolated,and the degree of pathological damage was observed by HE staining.Western blot and q RT-PCR experiments were performed to analyze the correlation between the expression changes of lnc RNA TUG1,mi R-320,STAT3,VEGF,Wnt-5a and?-catenin and changes in lower limb blood flow and lower limb ischemic pathology.Results:Lower extremity blood flow results: Compared with the control group,lower extremity blood flow velocities significantly decreased at 6h,12 h,24h,and 48 h after ischemia.HE staining results: Compared with the control group,the gastrocnemius muscle cells did not change significantly during 6h to 12 h of ischemia,but within12 h,24h,and 48 h,as the ischemic time prolonged,cytoplasmic eosinophilia gradually increased and cell necrosis increased.The gastrocnemius q RT-PCR results showed that compared with the control group,the activation of lnc RNA TUG1 gene gradually decreased and the expression of mi R-320 gene gradually increased with the prolonged ischemic time.Western blot results showed that compared with the control group,the expression of STAT3,VEGF,Wnt-5a and ?-catenin gradually decreased with the prolonged ischemic time.Conclusion:Within 48 hours of ischemia,as the ischemic time increased,the gastrocnemius injury gradually increased,which may be related to the inhibition of lnc RNA TUG1 expression and the promotion of mi R-320 expression.Part 2 Identification and cultivation of EPCsObjective:The proliferation,differentiation,migration and angiogenic ability of bone marrow-derived EPCs in nude mice were studied in vitro.Methods:Nude mice were taken off the neck to death,and femur and tibia were removed;the bone marrow was washed with culture medium,the washing fluid was collected,and cells were cultured with special medium for EPCs.After 14 days,cells were collected for flow cytometric identification,and CCK-8,Transwell,and Matrigel were used to detect EPCs proliferation,migration ability,and angiogenesis ability.Results:Nude bone marrow-derived cells had a CD14 positive rate of 99.9%,a CD34 positive rate of 86.4%,and a KDR positive rate of 0.1%.EPCs gradually proliferated within 1-5 days;and the number of migrating cells at 48 h was greater than 24h;Some EPCs can be seen to form clusters at 24 h,and more clusters at 48 h.Conclusion:Nude mouse bone marrow-derived cells contain EPCs,and they have the ability to grow,proliferate,migrate,and form blood vessels.Part 3 Effects of EPCs transplantation on lower limb ischemia in nude miceObjective:To investigate the changes of lnc RNA TUG1 and mi R-320 after EPCs transplantation to improve lower limb ischemia.Methods:Ten 6-week-old nude mice were randomly divided into 2 groups,5 in each group:(1)48h ischemia + EPCs group;(2)48h ischemia + NS group.Each group was fed with feed for 1 week,and nude mice were monitored to establish a model of lower limb ischemia in nude mice.Laser Doppler was used to detect the hemodynamic changes at 2d,5d,and 9d after ischemia.The lower limbs were dissected after 9th day,gastrocnemius muscles were isolated,the degree of pathological damage was observed with HE staining,and the microvessel density of gastrocnemius muscle was detected by immunohistochemistry.Western blot and q RT-PCR experiments were performed to analyze the correlation between the expression changes of lncRNA TUG1,mi R-320,STAT3,VEGF,Wnt-5a and?-catenin and changes in lower limb blood flow and lower limb ischemic pathology.Results:Lower extremity blood flow results: Compared with the control,the lower extremity blood flow of nude mice injected with EPCs recovered better on the 5th day after 48 hours of ischemia compared with the NS injection group.HE staining results: Compared with the control group,the lower limbs of the EPCs-injected group had lower eosinophilicity and cell necrosis in the gastrocnemius muscle cells compared to the NS-injected group.Immunohistochemical results: Compared with the control group,the gastrocnemius muscle injected with EPCs increased the microvessel density in the NS-injected group more.The gastrocnemius q RT-PCR results showed that compared with the control group,the gastrocnemius muscle injected with EPCs increased the expression of lnc RNA TUG1 gene and the mi R-320 gene expression compared with the NS group.Western blot results showed that compared with the control group,the gastrocnemius muscle injected with EPCs increased the expression of STAT3,VEGF,Wnt-5a and ?-catenin proteins compared with NS injected.Conclusion:After 48 hours of ischemia,EPCs could improve the ischemia of gastrocnemius muscle in nude mice,which might be related to promoting the expression of tug1 and inhibiting the expression of mir-320. |
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