大鼠脊髓神经元缺氧复氧损伤中钙敏感受体对其凋亡的影响及意义

摘要

目的 探究大鼠脊髓神经元缺氧复氧损伤中钙敏感受体对其凋亡的影响及意义.方法 将新生SD大鼠脊髓神经元随机分为正常对照组、缺氧复氧组、激动剂(GdCl3)组和抑制剂(NPS-2390)组4组.采用95％氮气通入置有细胞培养板的密闭通气盒中15 min,37℃培养箱内培养1h,再用含2％ B27、10％胎牛血清的Neurobasol-A液培养细胞24h复制缺氧复氧模型.应用免疫荧光技术鉴定脊髓神经元并观察钙敏感受体的表达定位,Westem blot法检测各组钙敏感受体及凋亡相关蛋白Caspase-3、Bax、Bcl-2的表达水平,激光共聚焦显微镜测定细胞内游离钙的变化,Tunel法观察各组细胞凋亡情况.结果 缺氧复氧组的钙敏感受体(=5.462,P=0.006)、细胞内游离钙(t=8.573,P=0.001)、细胞凋亡(t =4.899,P=0.008)、Caspase-3(t=5.118,P=0.007)和Bax (t=10.930,P=0.001)表达水平均明显高于对照组,激动剂组的钙敏感受体(t=4.975,P=0.008)、细胞内游离钙(t=4.899,P=0.008)、细胞凋亡(=7.746,P=0.002)、Caspase-3(=4.776,P=0.009)和Bax(t=5.281,P=0.006)表达水平均明显高于缺氧复氧组,抑制剂组的钙敏感受体(=3.674,P=0.021)、细胞内游离钙(t=3.846,P=0.018)、细胞凋亡(=4.281,P=0.013)、Caspase-3(t=3.521,P=0.024)和Bax(t=3.473,P=0.026)表达水平均明显低于缺氧复氧组;缺氧复氧组的Bcl-2表达水平明显低于对照组(t=6.242,P=0.003),激动剂组明显低于缺氧复氧组(t=3.028,P=0.004),抑制剂组明显高于缺氧复氧组(t=2.840,P=0.047).结论 在缺氧复氧损伤过程中,钙敏感受体表达增多,细胞内游离钙增加,脊髓神经元凋亡增多.%Objective To investigate the effect and significance of calcium-sensing receptor (CaSR) on the apoptosis of rat spinal cord neurons in anoxia/reoxygenation (A/R) injury.Methods The spinal cells were in ischemia and hypoxia environment for 1 h and in normal environment for 24 h to establish a model of A/ R.After spinal A/R model was established,the spinal cells were divided into four groups randomly:the control group,A/R group,A/R + GdCl3 group,and A/R + NPS-2390 group.The expression of CaSR in each group was detected by immunofluorescence and Western blotting.The concentration of intracellular calcium was measured by laser confocal scanning microscopy.The expressions of Caspase-3,Bax,and Bcl-2 were detected by using Western blotting.The apoptotic rate of spinal cells was detected by Tunel assay.Results Compared to the control group,there was a significant increase in the level of CaSR (t =5.462,P =0.006),the concentration of intracellular calcium (t =8.573,P =0.001),the apoptotic rate (t =4.899,P =0.008),Caspase-3 (t =5.118,P =0.007),and Bax (t =10.930,P=0.001) in A/R group.Compared to the A/R group,there was a significant increase in the level of CaSR (t =4.975,P =0.008),the concentration of intracellular calcium (t =4.899,P =0.008),the apoptotic rate (t =7.746,P =0.002),Caspase-3 (t =4.776,P =0.009),and Bax (t =5.281,P =0.006) in A/R + GdC13 group.Compared to the A/R group,there was a significant decrease in the level of CaSR (t =3.674,P =0.021),the concentration of intracellular calcium (t =3.846,P =0.018),the apoptotic rate (t =4.281,P=0.013),Caspase-3 (t =3.521,P=0.024),and Bax (t =3.473,P =0.026) in A/R + NPS-2390 group.However,compared to the control group,there was a significant decrease in the level of Bcl-2 (t =6.242,P =0.003) in A/R group.Compared to the A/R group,there was a significant decrease in the level of Bcl-2 (t =3.028,P =0.004) in A/R + GdCl3 group.Compared to the A/R group,there was a significant increase in the level of Bcl-2 (t =2.840,P =0.047) in A/R + NPS-2390 group.Conclusion During the process of A/R injury in rat spinal cord neurons,the expression of calcium sensing receptor increases,along with increase in intracellular calcium and spinal neuron apoptosis.