【摘要】 目的:探讨Egr-1基因在吗啡慢性处理或吗啡戒断细胞中的表达以及在吗啡成瘾过程中的作用。方法:分别用浓度为0、1、10、100和1000μmol/L的吗啡和0、0.01、0.1、1和10μmol/L纳洛酮急性处理PC12细胞,1h后Egr-1蛋白免疫荧光染色。吗啡慢性处理实验分4组,A组,B组、C组和D组。C组和D组细胞加入100μmol/L吗啡,培养48h,A组和B组仅加入同体积0.9%氯化钠。48h后B组和D组给予10μmol/L纳洛酮拮抗,A组和C组加入同体积0.9%氯化钠。在纳洛酮拮抗后1、2、4、8和24h,Egr-1蛋白免疫荧光染色;用MTT比色法观察各组PC12细胞的损害,并用FITC-AnnexinⅤ/PI流式凋亡检测试剂盒检测细胞凋亡率。结果:0~100μmol/L吗啡及各浓度的纳洛酮作用下细胞未见明显Egr-1阳性染色,而1000μmol/L时有少量阳性染色细胞。D组在纳洛酮戒断后1 h即出现Egr-1蛋白阳性染色并持续24 h,并在12 h时可见部分细胞折光性能力减弱,细胞中颗粒增多,核固缩并断裂成数个等。C组仅有少许细胞出现Egr-1阳性染色。与A组相比较,C组细胞生存率下降,并在12 h和24 h时差异有统计学意义;而D组生存率则进一步下降,与A组和C组相比差异有统计学意义。FITC-AnnexinⅤ/PI流式凋亡检测到C组细胞凋亡8.42±2.09%,D组细胞凋亡率则增加到37.62±7.19%。结论:吗啡慢性处理或吗啡慢戒断后出现Egr-1基因表达增加和细胞凋亡,而Egr-1基因的表达增加和细胞凋亡可能参与吗啡成瘾机制。更多还原

【Abstract】 Objective: To study the expression and effect of Egr-1 gene in PC12 cells after chronic morphine treatment or morphine withdrawal.Methods: In the experiments testing whether acute morphine treatment can induce Egr-1 protein expression,the PC12 cells were treated with 0～1000 μmol/L of morphine and 0～10 μmol/L naloxone.After the cells were treated with 100μmol/ L morphine for 48 h,10μmol/ L naloxone was used to produce acute withdrawal.EGR-1 protein was detected by immunofluorescence.PC12 cells were treated with morphine for 48 h before acute withdrawal with naloxone,and then thiazolyl blue tetrazolium bromide(MTT) assays were used to detect the proliferation of the cells and cells apoptosis were determined by AnnexinⅤ/PI flow cytometry.Results: A little EGR-1 protein staining was observed at 1000 μM morphine and chronic morphine treatment.The forms of cells were irregular and spindle,the chromatin concentrated into masses,the apoptosis bodies were formed 24h after morphine withdrawal.EGR-1 protein expression was appeared at 1h after withdrawal,reached peak at 8h and was detected even at 24h.MTT assays showed that mortality declined significantly(P<0.05),and the percentage of apoptosis of PC12 cells after chronic morphine treatment and morphine withdrawal are 8.42%±2.09% and 37.62%±7.19% respectively.Conclusion: The expression of Egr-1 gene was increased and apoptosis appeared in PC12 cells after chronic morphine treatment or morphine withdrawal,which might play a role in opioid addiction and may be the response of morphine withdrawal.更多还原