| Part I TO ESTABLISH AN ANIMAL MODEL ON SPINAL CORD CELLS APOPTOSISObjective:Selective transection unilateral sciatic nerve,the model of spinal cord cell apoptosis of the spinal cord was constructed.Methods:The right side of the sciatic nerve in neonatal rats was cut off above the branch of sciatic nerve,using the sponge with sterile NS to surround the proximal severed sciatic nerve,establishing the model of spinal cord cell apoptosis model of the spinal cord.Using various methods(TUNEL immunofluorescence、Quantitative Real-time PCR and western blot)to observe the morphological changes at different time points.Results:1.Comparing the model group with the control group after sciatic nerve transected 6h,12 h,24h,48 h,TUNEL immunofluorescence staining results showed that the difference of cell apoptosis number in the spinal cord was statistically significant(P<0.05).Among these time points,24 h have the largest number of TUNEL positive cells.2.Data analysis by Quantitative Real-time PCR experiments indicated that the expression level of Caspase-3 gradually increased and peaked at 24 h after sciatic nerve injury.The expression level of Bax and Bcl-2 increased with time after sciatic nerve injury,the expression level of Bax peaked at 12 h,while Bcl-2 peaked at 24 h.The ratio of Bcl-2/Bax reaches the minimum at 12 h and 24 h.The transcription level of genes which promote apoptosis: Caspase-3 and Bax increased evidently at different time points(6h,12 h,24h,48h)after sciatic nerve transected,the difference was statistically significant compared with sham group(P<0.05).3.The result of western blot was consistent with the result of Quantitative Real-time PCR experiments.The protein expression of Caspase-3 and Bax increased obviously at different time points(6h,12 h,24h,48h)after injury,while the expression of Bcl-2 increased slightly.It indicated that the rat model of spinal cord cell apoptosis was builded.successfullyConclusion:The model of cell apoptosis in spinal cord and dorsal root ganglion was established successfully.Part II EFFECTS OF ACHYRANTHES BIDENTATA POLYPEPTIDES ON APOPTOSIS IN SPINAL CORD AND DORSAL ROOT GANGLION INDUCED BY UNILATERAL SCIATIC NERVE TRANSECTIONObjective: To detect the effects of Achyranthes Bidentata Polypeptides onapoptosis in spinal cord and dorsal root ganglion induced by unilateral sciatic nerve injury.To explore the role and the mechanim of Achyranthes Bidentata Polypeptides on cell apoptosis of the spinal cord and dorsal root ganglion,which may provide certain theoretical basis and experimental evidence for the treatment of spinal cord injury.Methods:1.The rats born one day for research of our experiment.On the proximal of transected sciatic nerve were given 10μl of normal saline(NS),nerve growth factor-β(NGF-β)and different concentrations of Achyranthes Bidentata Polypeptides(0.001mg/ml,0.01mg/ml,0.1mg/ml,1mg/ml),therefore the subjects were divided into sham surgery group 、 negative control group 、 positive control group and experimental group.2.Using the method TUNEL immunofluorescence to observe the cell apoptosis of spinal cord and dorsal root ganglion in intumescentia lumbalis.3.Quantitative Real-time PCR was applied to detected the expression of mRNA level of Caspase-3、Bcl-2、Bax.4.Western blot was used to detect the protein expression level of Caspase-3,Bcl-2 and Bax protein.Results:1.TUNEL immunofluorescence results showed that after given Achyranthe s Bidentata Polypeptides(0.01mg/ml,0.1mg/ml,1mg/ml)the number of TUNEL positive cells in spinal cord and dorsal root ganglion was reduced compared with NS group,the difference was statistically significant(P<0.05).2.With different concentrations of Achyranthes Bidentata Polypeptides administered after sciatic nerve transected,the result of Quantitative Real-time PCR indicated that the expression of Caspase-3 and Bax mRNA in experiment group decreased to different degree comapred with NS group.The expression of Caspase-3and Bax mRNA in Achyranthes Bidentata Polypeptides with 0.1mg/ml and1 mg/ml group was reduced significantly compared with NS group,the difference has statistical significance(P<0.05).But compared with the control group and NGF-βgroup there had no statistical significance(P>0.05).The expression of anti-apoptotic gene of Bcl-2 was increased,and it was increased more significantly by giving0.1mg/ml and 1mg/ml of Achyranthes Bidentata Polypeptides,the ratio of the Bcl-2/Bax also have the same change.3.The results of western blot showed that the expression of promoting apoptosis protein Caspase-3、Bax had decreased,while the expression of anti-apoptosis Bcl-2protein was increased after given Achyranthes Bidentata Polypeptides.Comparing Achyranthes Bidentata Polypeptides group(concentration of 0.1mg/ml,1mg/ml)with NGF-β group the expression of three apoptosis related factors had no statistical significance(P>0.05).And the analysis showed that the ratio of bcl-2/bax was highestby giving the concentrations of Achyranthes Bidentata Polypeptides were 0.1 mg/ml or 1 mg/ml.Conclusions: Achyranthes Bidentata Polypeptides can inhibit the cell apoptosis in spinal cord and dorsal root ganglion induced by unilateral sciatic nerve transection.And it has nerve protective effect.This protection mainly by downregulating the expression of pro-apoptotic gene(Caspase-3 and Bax)which promote apoptosis,and upregulating the expression of anti-apoptotic gene(Bcl-2)to decrease spinal cord and dorsal root ganglion cell apoptosis induced by sciatic nerve transection. |
