【作者】 何炜；

【导师】 黄永辉；

【作者基本信息】 江苏大学 ， 外科学（专业学位）， 2017， 硕士

【摘要】 目的:探究钙敏感受体(Calcium-sensing Receptor,CaSR)在大鼠脊髓神经元中的表达情况,进一步研究CaSR对大鼠脊髓神经元缺氧复氧(anoxia/reoxygenation,A/R)损伤的影响及机制,为治疗脊髓缺血再灌注损伤(spinal cord ischemia reperfusion injury,SCIRI)指出新的研究方向及治疗靶点。方法:1.从健康新生SD大鼠中提取脊髓神经元,应用免疫荧光技术鉴定脊髓神经元并观察CaSR在脊髓神经元中的表达定位。2.建立大鼠脊髓神经元缺氧复氧损伤模型并评价。3.应用Gd Cl3(CaSR激动剂)、NPS-2390(CaSR抑制剂),将脊髓神经元随机分成4组:正常对照(control)组、缺氧/复氧(A/R)组、A/R+Gd Cl3组、A/R+NPS-2390组。采用免疫印迹法(Western-blotting)检测各组CaSR蛋白表达水平;应用荧光定量PCR技术检测各组CaSR m RNA的表达。应用激光共聚焦显微镜(Laser Confocal Scanning Microscope,LCSM)测定细胞内游离钙浓度([Ca2+])的变化,同时观察Gd Cl3和NPS-2390对细胞内游离钙的影响。4.采用原位末端标记法(Td T-mediated biotinylated-d UTP nick-end labeling,Tunel)观察各组细胞凋亡情况,并用Western-blotting检测凋亡相关蛋白Caspase-3、Bax和Bcl-2的表达。结果:1.CaSR在大鼠脊髓神经元中有表达,并且主要分布在神经元胞膜和胞浆中。2.大鼠脊髓神经元缺氧复氧损伤模型建立成功,模型组的CaSR蛋白较对照组表达高。3.模拟脊髓神经元缺氧复氧损伤后CaSR蛋白、CaSR m RNA表达明显增加,Gd Cl3能够使缺氧复氧损伤所诱发的CaSR蛋白、CaSR m RNA表达进一步增加,而NPS-2390的作用则相反。模拟缺氧复氧损伤后细胞内游离钙表达明显增加,Gd Cl3能够使缺氧复氧损伤所诱发的细胞内游离钙表达进一步增加,而NPS-2390的作用则相反。4.模拟脊髓神经元缺氧复氧损伤后凋亡相关蛋白Caspase-3、Bax蛋白表达进一步增加,Bcl-2蛋白表达进一步减少,脊髓神经元凋亡加重;Gd Cl3组进一步加重损伤,而NPS-2390的作用则相反。结论:CaSR在大鼠脊髓神经元中有广泛的表达,并且主要分布在神经元胞膜和胞浆中。建立大鼠脊髓神经元缺氧复氧损伤模型后可发现CaSR表达增加,细胞内游离钙增加,脊髓神经元凋亡增多,CaSR激动剂Gd Cl3可使CaSR表达进一步增加,加重脊髓神经元损伤,而CaSR抑制剂NPS-2390可使CaSR表达减少,减轻脊髓神经元的损伤。提示临床上脊髓缺血再灌注损伤中对CaSR的干预可成为治疗疾病的新思路。更多还原

【Abstract】 Objective:To investigate the expression of calcium-sensing receptor(CaSR)in spinal cord neurons and to study the effect and significance of CaSR in spinal cord neurons anoxia/reoxygenation(A/R)injury,in order to point out a new research direction and therapeutic targets.Methods:1.Spinal cord neurons were obtained from the SD rats.Using immunofluorescence technique to identify the spinal cord neurons and observe the expression of CaSR in spinal cord neurons.2.To establish the model of spinal cord neurons anoxia/reoxygenation(A/R)injury.3.The spinal cells were divided into four groups randomly:the control group,anoxia/reoxygenation group(A/R group),A/R+GdCl3 group and A/R+NPS-2390 group.The expression of CaSR in each group was detected by Western-blotting and Fluorescent quantitative PCR.The concentration of intracellular calcium was measured by laser confocal scanning microscope while observing the effection of intracellular calcium by GdCl3 and NPS-2390.4.The apoptotic rate of spinal cells was detected by Tunel assay.Using Western-blotting to detect the expression of Caspase-3,Bax and Bcl-2.Results:1.The expression of CaSR was existed in rat spinal cord neurons and distributed in the cell membrane and cytoplasm.2.The model of spinal cord neurons anoxia/reoxygenation(A/R)injury was established successfully.The expression of CaSR protein in A/R group was higher than in control group.3.Compared to the control group,there was a significant increase in the levels of CaSR protein,CaSR mRNA and the concentration of intracellular calcium in A/Rgroup.However,the above parameters increased more in A/R+GdCl3 group,and decreased in A/R+NPS-2390 group.4.Compared to the control group,there was a significant increase in the levels of Caspase-3,Bax,and the apoptotic rate in A/R group,accompanied with the decreasing of Bcl-2.However,the above parameters increased more in A/R+GdCl3 group,and decreased in A/R+NPS-2390 group.Conclusions:The expression of CaSR was existed in rat spinal cord neurons and distributed in the cell membrane and cytoplasm.During the process of anoxia/reoxygenation injury in rat spinal cord neurons,the expression of calcium sensing receptor and calcium is increased,accompanied with the increasing apoptosis rate.GdCl3 can increase the expression of CaSR and make spinal cord neurons anoxia/reoxygenation injury deeper.However,NPS-2390 has the opposite effect.It is suggested that the intervention of CaSR in spinal cord ischemia-reperfusion injury can be a new way to treat the disease.更多还原