卒中后抑郁患者多巴胺代谢系统COMT和DAT基因多态性分析

目的 探讨多巴胺递质代谢相关蛋白儿茶酚氧位甲基转移酶(COMT)Val108/158Met、多巴胺转运体(DAT)40bp可变数目串联重复多态(VNTR)两位点基因多态性与卒中后抑郁(PSD)的关系.方法 选择南方医科大学珠江医院神经内科自2010年1月至2010年6月收治的PSD患者68例作为研究组,同期脑卒中后无抑郁患者91例作为对照组,利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术分析患者COMT Val108/158Met、DAT 40 bp VNTR位点的基因多态性.结果 COMT基因扩增产物分野生型(G/G)、突变纯合型(A/A)、杂合型(A/G)3种基因型,DAT基因扩增产物分7/7、9/7、10/7、10/9、10/10、11/10、11/11倍重复7种基因型;2组患者COMT等位基因、基因型分布比较差异均有统计学意义(x2=5.703,P=0.017;x2=6.489,P=0.039).研究组男性患者与对照组男性患者之间比较,COMT等位基因、基因型的分布差异均无统计学意义(P＞0.05);研究组女性患者与对照组女性患者之间比较,COMT等位基因、基因型的分布差异均有统计学意义(x2=4.610,P=0.032;x2=6.547,P=0.024);研究组与对照组患者DAT等位基因和基因型的总体分布、杂合度比较差异均无统计学意义(P＞0.05).结论 COMT Val108/158Met基因多态性与PSD发生可能有相关性,而DAT40 bp VNTR基因多态性与PSD发生无明显相关性.

Abstract：

Objective To investigate the association of post-stroke depression (PSD) with gene polymorphisms of catechol-O-methyl transferase (COMT) Val1 08/158Met and dopamine transporter 40bp variable number of tandem repeats (VNTR) in dopamine metabolism system. Methods Sixty-eight patients with PSD and 91 patients only suffered from stroke, admitted to our hospital from January 2010to June 2010, were chosen; the gene polymorphisms ofCOMT Val108/158Met and DAT 40 bp VNTR were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results The genotypes of COMT gene amplifications were wild type (G/G),homozygous mutant type (A/A) and heterozygous type (A/G); 7 repeated genotypes (7/7, 9/7, 10/7, 10/9,10/10, 11/10 and 11/11) were noted in the DA T gene amplifications; frequencies of COMT alleles and genotypes were significantly different between the 2 groups (x2=5.703, P=0.017;x2=6.489, P=0.039). The frequencies of COMT alleles and genotypes were significantly different between the 2 female groups (x2=4.610, P=0.032;x2=6.547, P=0.024), but no significant differences were found between the 2 male groups (P＞0.05). The frequencies and heterozygosity of DAT alleles and genotypes showed no obvious differences between the 2 groups (P＞0.05). Conclusion The gene polymorphism of COMT Val108/158Met may be associated with PSD, while that of DAT 40bp VNTR is not.     

Hallervorden-Spatz综合征的临床、影像学特点及脑深部电刺激治疗

目的 分析Hallervorden-Spatz综合征(HSS)的临床、影像学特点及脑深部电刺激术(DBS)治疗后效果. 方法 回顾性分析南方医科大学珠江医院于2010年6月收治的l例HSS患者的临床、影像学特点及DBS治疗后预后. 结果 本例患者男性,12岁,发病年龄较早,临床以肌张力障碍、智能障碍、构音困难、锥体束受累为主要症状,病程进展较快;头颅MRI表现为典型的“虎眼征”；采用DBS[刺激靶点为双侧苍白球内侧部]治疗后半年患者运动症状明显改善,但言语及智能改善不明显,随访1年患者病情平稳. 结论 典型影像学特点和临床症状是临床诊断HSS的重要依据,DBS能改善HSS患者的运动症状,是该病是有限的可选择治疗手段之一.     

JAK/STAT信号转导通路在IGF-1抑制左旋多巴诱导的多巴胺能神经元凋亡中的作用

目的 探讨胰岛素样生长因子1(IGF-1)通过JAK/STAT信号转导通路对左旋多巴诱导的多巴胺能神经元凋亡的保护作用.方法 应用100μg/L β神经生长因子(β-NGF)将体外常规培养的PC12细胞诱导分化为多巴胺能神经元.MTT比色法筛选左旋多巴的神经损伤浓度和IGF-1的神经保护浓度(150μmol/L、100nmol/L),实验分为4组:(1)左旋多巴+IGF-1组;(2)左旋多巴+IGF-1+AG490组,AG490(10 μmol/L)在给予左旋多巴和IGF-1前20 min加人;(3)左旋多巴组;(4)对照组.Western blotting检测4组细胞P-JAK2、P-STAT3蛋白表达,Hoechst33258染色和流式细胞仪分别检测细胞凋亡和凋亡率.结果 Westen blotting法检测显示对照组和左旋多巴组P-JAK2、P-STAT3蛋白表达阴性,而左旋多巴+IGF-1组和左旋多巴+IGF-1+AG490组表达阳性.左旋多巴+IGF.1+AG490组P.JAK2、P.STAT3表达强度低于左旋多巴+IGF.1组,差异有统计学意义(P<0.05):Hoechst33258染色结果显示左旋多巴组细胞核固缩及碎裂最明显,较多凋亡小体形成,而左旋多巴+IGF-1组与左旋多巴+IGF-1+AG490组仅有少量凋亡小体形成.流式细胞仪检测显示4组多巴胺能神经元凋亡率不同,差异有统计学意义(F=180.991,P=0.000);与对照组比较,另3组细胞凋亡率均增高,差异有统计学意义(P<0.05);其中左旋多巴组细胞凋亡率最高,其次为左旋多巴+IGF-1+AG490组、左旋多巴+IGF-1组.结论 大剂量左旋多巴对多巴胺能神经元具有毒性作用.IGF-1对左旋多巴诱导的神经细胞毒性作用呈保护作用,JAK2/STAT3信号转导通路参与了此过程.

Abstract：

Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.     

JAK/STAT信号转导通路在IGF-1抑制左旋多巴诱导的多巴胺能神经元凋亡中的作用

Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.     

JAK/STAT信号转导通路在IGF-1抑制左旋多巴诱导的多巴胺能神经元凋亡中的作用

Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.     

SIRT1在脂多糖诱导的PC12细胞凋亡中的作用

目的 探讨沉默信息调控因子1(SIRTl)在脂多糖(LPS)诱导的PC12细胞凋亡过程中的作用. 方法 PC12细胞按不同培养方法分为6组:对照组、250 μg/mL组、500 μg/mL组、750μg/mL组、1000 μg/mL组、1250 μg/mL组;对照组常规培养,后5组添加相应浓度的LPS培养.24 h后MTT法测量细胞存活率,同时确定LPS诱导PC12细胞凋亡的适合浓度.再按选定的浓度培养细胞,并根据培养时间的不同分为6组:对照组、1/2 h组、2h组、18h组、24 h组、48 h组,Hoechst染色及流式细胞仪测量细胞凋亡率.Western blotting检测各组细胞中SIRT1的表达情况. 结果 Hoechst染色结果提示PCl2细胞经LPS培养1/2h后出现凋亡,表现为核固缩、核碎裂;18h开始凋亡小体增多,24h达到高峰,48 h后又有所下降.流式细胞仪检测结果显示各实验组细胞凋亡率与对照组相比差异均有统计学意义(P＜0.05),凋亡率变化趋势与Hoechst染色法观察到的结果 一致.Western blotting结果显示对照组SIRTl表达量为1.84±0.04; 1/2 h时SIRT1蛋白表达减低至1.17±0.09;24 h时表达降至最低,为0.62±0.03;48 h组表达量有所回升,达到0.77±0.02;实验组各时间点组与对照组相比差异均有统计学意义(P＜0.05). 结论 LPS可以诱导PC12细胞凋亡,SIRT1在此过程中的表达受到抑制;推测SIRT1在LPS诱导PC12细胞凋亡的过程中起到一定的保护作用.