| Objective:(1) To explore the transfection efficiency of that the inhibitory polypeptide of Rho-ROCKII was introduced into dorsal root ganglia neurons(DRGNs) in vitro with Lipofectamine 2000 and the optimized factors affecting the transfection efficiency of liposome. (2) To observe the effect on DRGNs axonal growth and extension with application of Inhibitory polypeptide of Rho-ROCKII in vitro. (3) To Compare the effect on DRGNs axonal growth and extension of rats treated with Y27632 and the micromolecule polypeptide. Methods:(1) 10 adult female Sprague-Dawley (SD) rats were randomly selected into a groups and were subjected to weight-drop impact causing complete paraplegia, The T8-10 spinal cord extracts (SCE) were harvested in complete paraplegia group at 7th day after spinal cord injury in rats. (2) All thoracic-lumbar DRGNs of neogenic SD rats (<5d) were harvested, and then DRNGs were harvested, indentified, Cultured, purified and amplified. (3) Based on the most optimized transfection condition (the ratio of liposome and polypeptide is 2.5:1), The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed at different concerations of liposome/inhibitory polypeptide respectively. (4)The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed in vitro after co-culture with complete paralysis SCE+ Y26732 or liposome/ inhibitory polypeptide at 2 days respectively. Result:(1)The mean lengths of neuronal axons of neogenesis DRGNs was 197±14um in group A, 301±17um in group B,316±25um in group C,367±29um in group D,265±16um in group E and291±21um in group F respectively at 2 days after co-culture. The axonal length in group D was significantly longer compared to group A, B, C and E respectively.there was no difference among group A,B, C and E. The mean fluorescence densities at axonal distal end were 197.8±20.1 AFU/um in group A,231.8±19.1AFU/um in group B,249.8±22.8 AFU/um in group C,289.1±17.1 AFU/um in group D,269.8±21.6AFU/um in group E and 271.6.8±16.2 AFU/um in group F respecviely at 2 days after co-culture.The mean fluorescence densities in group D was significantly stronger than it in group A,B, C and E and there is no significant difference among group A,B, C and E. (2) The mean lengths of neuronal axons of neogenesis DRGNs was391±20um in group A (DRGNs+PBS),107±21 um in group B (DRGNs+ complete paralysis SCE),121±23um in group C (DRGNs+complete paralysis SCE+liposome),307±16um in group D (DRGNs+complete paralysis SCE+Y26732) and 367±19um in group E (DRGNs+complete paralysis SCE+liposome+inhibitory polypeptide)respectively at 2 days after co-culture. The axonal length in group B and group C were shorter than it in group A,D,and E, and group E was longer than group D, there was no difference between the groups B and C. The mean fluorescence densities at axonal distal end were 183.4±3.7AFU/um in group A,56.1±4.2AFU/um in group B, 62.7±5.9 AFU/um in group C,278.1±6.2AFU/um in group D and 313.4±3.6AFU/um in group E in group F respecviely at 2 days after co-culture. The mean fluorescence densities at axonal distal end in groupA,D,E were stronger than groupB, C and group E was stronger than group A,D, there was no difference between the groups B and C. Conclusion:(1) The inhibitory polypeptide of Rho-ROCKⅡtransfectd by Lipofectamine 2000 can promote axon extend, induce regeneration of axon branches. The highest efficiency was achieved in the condition of 8ug polypeptide and 20μl liposome. (2) The effect of promoting axon extend of the inhibitory polypeptide is significantly stronger than Y27632. |
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